Reviews

Microbiome risk profiles as

biomarkers for inflammatory
and metabolic disorders
Amira Metwaly 1
, Sandra Reitmeier2 and Dirk Haller 1,2 ✉

Abstract | The intestine harbours a complex array of microorganisms collectively known as the
gut microbiota. The past two decades have witnessed increasing interest in studying the gut
microbiota in health and disease, largely driven by rapid innovation in high-​throughput multi-
omics technologies. As a result, microbial dysbiosis has been linked to many human pathologies,
including type 2 diabetes mellitus and inflammatory bowel disease. Integrated analyses of multi-
omics data, including metagenomics and metabolomics along with measurements of host
response and cataloguing of bacterial isolates, have identified many bacteria and bacterial
products that are correlated with disease. Nevertheless, insight into the mechanisms through
which microbes affect intestinal health requires going beyond correlation to causation. Current
understanding of the contribution of the gut microbiota to disease causality remains limited,
largely owing to the heterogeneity of microbial community structures, interindividual differences
in disease evolution and incomplete understanding of the mechanisms that integrate microbiota-
derived signals into host signalling pathways. In this Review, we provide a broad insight into the
microbiome signatures linked to inflammatory and metabolic disorders, discuss outstanding
challenges in this field and propose applications of multi-​omics technologies that could lead
to an improved mechanistic understanding of microorganism–host interactions.

Microbiome
All the microorganisms, their
genomes and the surrounding
host-​shaped environmental
conditions of a given habitat.
The gut microbiome can be
characterized through the
application of metagenomics,
metabolomics, metatranscrip-
tomics and metaproteomics
combined with clinical or
environmental metadata.
1
Chair of Nutrition and
Immunology, Technical
University of Munich,
Freising, Germany.
2
ZIEL Institute for Food &
Health, Technical University of
Munich, Freising, Germany.
✉e-​mail: dirk.haller@tum.de
https://doi.org/10.1038/
s41575-022-00581-2
The human digestive tract harbours a complex array of
microorganisms, including bacteria, archaea, viruses
and fungi. Trillions of bacteria colonize the gastroin-
testinal tract in a spatially structured manner and their
combined genomes contain more than 200 times the
number of genes in the human genome. Colonization
density exhibits a gradient from the proximal to the
distal part of the gut, such that the highest numbers of
microorganisms are found in the colon1,2. In contrast to
the rapid transit of material through the small intestine
(3–6 h), the comparatively reduced motility of the colon
(20–50 h) results in retention of luminal content, which
builds a reservoir of biologically active metabolites.
In addition to microorganisms, the term microbiome
encompasses the environment or niche inhabited by the
microbiota, which is shaped by the host. The incorpora-
tion of host-​related factors provides a broad view of the
gut ecosystem, in which bidirectional microorganism–
host interactions influence the physical and chemical
characteristics of the microbial environment or theatre
of activity3. As the digestive tract and its microbiome
are considered to lie at the intersection of immune
and metabolic processes, this Review focuses on
inflammatory bowel disease (IBD) and type 2 diabetes
mellitus (T2DM) as exemplars of microbiota-​associated
disorders.
IBD and T2DM are both recognized to be multi-
factorial diseases with a global pattern of rising disease
incidence following industrialization4–6. Their aetiology
involves a complex interplay of genetic susceptibil-
ity, environmental triggers and urban lifestyle-​related
factors. Against this shared background, metabolic dis-
eases (such as T2DM) are additionally characterized by
chronic subclinical inflammation in the liver, adipose
tissue, muscles, pancreas and gut, whereas inflamma-
tory gastrointestinal disorders, such as Crohn’s disease
(CD) and ulcerative colitis (UC), are additionally asso-
ciated with inflammation-​driven metabolic alterations7.
Genome-​wide association studies have identified large
numbers of genetic variants associated with increased
susceptibility to developing either T2DM (143 loci)8 or
IBD (>240 loci)9. However, these variants collectively
explain only a small proportion of the heritability of
these disorders: <10% for T2DM, <15% for UC and
<50% for CD10–15. This situation suggests the impor-
tance of environmental triggers, and in particular the
gut microbiome, as major contributors to the aetiology
of these diseases.

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Key points
• Several commonalities exist between inflammatory bowel disease (IBD) and type 2
diabetes mellitus (T2DM), two multifactorial diseases that show a pattern of
increasing global incidence following industrialization.
• Altered gut bacterial composition and changes in host processing of bacteria-​derived
metabolites are implicated in IBD and T2DM and provide a shared underlying
pathogenetic mechanism.
• A causal link between dysbiotic microbial communities and IBD or T2DM has been
established through gnotobiotic mouse experiments and integrative multi-​omics
studies.
• Challenges in disease-​specific biomarker discovery include determining the causality
of observed changes, understanding their functional redundancy in disease
mechanisms and the geographic and ethnic heterogeneity of gut microbiota.
• Big data refinement, testing and validation of specific bacterial strains, their encoded
genes and metabolic byproducts are necessary to identify disease biomarkers.
Despite great advances in disease risk profiling driven
by data from genome-​wide association studies and
multi-​omics approaches, the identification of susceptible
individuals is still difficult and validated diagnostic or
prognostic markers are lacking. However, multiple anal-
yses of large population studies and cohorts of patients
with IBD or T2DM have identified microbiome signatures
linked to specific disease phenotypes16–19, the risk of
relapse20 and response to treatment21.
In this Review, we summarize current knowledge
on the involvement of the gut microbiome in IBD and
T2DM. We critically assess the status of currently avail-
able disease-​associated microbiome signatures and dis-
cuss the limitations that define their clinical use. Finally,
we discuss how ‘big data’ generated by multi-​omics
approaches and analysed in an integrative framework
can be used to disentangle the complex pathology of
these diseases. In particular, we focus on the mecha-
nisms underlying interactions between bacterial strains
and gut-​derived metabolites that promote processes
involved in inflammatory and metabolic diseases such
as IBD and T2DM.
Microbiota
The microorganisms (bacteria, Common features of IBD and T2DM
fungi, archaea and viruses)
of a defined environment.
Both IBD and T2DM are associated with characteris-
tic microbial alterations, notably reduced community
Microbiome signatures richness with the reduction of beneficial microbes and
Unique patterns of microbiome expansion of pathobionts22. The challenge in understand-
configuration that can stratify
ing the role of microbial alterations in disease initiation
defined physiological and
pathological conditions and and progression lies in the timing of such changes (that
can be used to predict the risk is, whether they are a cause or consequence of the dis-
of disease development or ease), the functional redundancy of some changes
progression. (meaning that they result in a similar signal or effect
Pathobionts
on disease-​related mechanisms) and the fluctuations in
Microorganisms that are linked these changes occurring during the disease course (such
to chronic inflammatory fluctuations can only be observed by longitudinal sam-
conditions but are harmless pling, which is rarely available). Despite the differences
to the host under normal
in their pathology, IBD and T2DM share several mech-
conditions.
anistic features. The metabolic features of T2DM are
Faecal microbiota accompanied by chronic low-​grade inflammation and
transplantation disruption of the gut barrier, and recurrent inflamma-
(FMT). A treatment based on tory flares in patients with IBD coevolve with metabolic
transfer of microorganisms
from the stool of a healthy
alterations at the cellular and systemic level23,24.
donor into a patient’s Therapy for these complex diseases remains challeng-
digestive system. ing, but controlled trials of faecal microbiota transplantation
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(FMT) have shown clinical efficacy in both T2DM25
and IBD26–30, including UC and, to a lesser extent,
CD. Clinical trials of FMT also provide evidence of a
causal relationship between the gut microbiota and
other inflammatory, immune or metabolic disorders31.
For example, FMT is highly effective in treating
approximately 90% of patients with Clostridioides
difficile (formerly Clostridium difficile) infection 32
and has been assessed as a treatment for obesity33 and
graft-​versus-​host disease34. In four randomized clin-
ical trials, FMT induced clinical remission in 28% of
patients with UC28,35–37. Few clinical trials have exam-
ined the efficacy of FMT in patients with CD and the
results have been rather heterogeneous. In one study of
174 patients with CD treated with FMT, clinical remis-
sion was achieved in 20% of patients and, overall, 43% of
patients achieved a clinical response38. A separate rand-
omized controlled trial found no effect of FMT on rates
of clinical remission in patients with CD, but increased
engraftment of donor microbiota was associated with
maintenance of remission27.
Conversely (and despite a multitude of studies
demonstrating that either dysbiosis specifically or par-
ticular microbiota profiles are linked to metabolic dis-
orders), evidence of a benefit of FMT in patients with
metabolic diseases is less well established. Landmark
studies demonstrated metabolic improvements as well
as beneficial changes in the intestinal microbiome in
patients with metabolic syndrome who received FMT
from lean, healthy donors29. However, these effects were
inconsistent and transient, which could be explained by
limited engraftment of the donor microbiota29 or by var-
iations in donor faecal microbial diversity at baseline25.
Intriguingly, supplementation with low-​fermentable
fibre following oral FMT led to improved insulin sen-
sitivity, increased microbial diversity and richness, and
prolonged donor microbial engraftment in patients with
both obesity and metabolic syndrome33. These data
emphasize the value of microbial modulation therapy
in reversing metabolic dysfunction. In line with these
findings, FMT from metabolically compromised donors
with obesity transiently worsened insulin sensitivity in
recipients with metabolic syndrome, whereas FMT from
healthy donors after gastric bypass induced a minimal
increase in insulin sensitivity in recipients with meta-
bolic syndrome39. This study provides evidence for the
transmissibility of donor metabolic profiles by FMT.
Microbial dysbiosis in IBD. Several large cohort
studies (Tables 1,2) have investigated gut microbiota
alterations in patients with IBD by profiling their
luminal and mucosal microbial communities. Overall,
active IBD is associated with an overabundance of
certain bacterial groups such as Enterobacteriaceae,
Fusobacterium, Ruminococcus gnavus, Streptococcus
anginosus, Enterococcus, Megasphaera, Campylobacter
and sulfate-​reducing Gammaproteobacteria and Delta­
proteobacteria. Conversely, IBD is linked to the loss
of beneficial taxa such as Faecalibacterium prausnitzii,
Christensenellaceae, Collinsella, Roseburia, Ruminococcus
and other butyrate-​producing bacteria17,20,21,40–44. Shotgun
metagenomics of stool samples has provided a more
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Table 1 | Gut microbiome signatures associated with IBD

Study population Methodology Risk signature Ref.
Increased Decreased
67 patients with IBD (34 CD, 33 UC); 16S rRNA gene Fusobacterium, Escherichia coli Faecalibacterium, 17

diagnoses confirmed by endoscopic sequencing of V4 (loss of beneficial microorganisms Peptostreptococcaceae,

and histological criteria, in remission region; multivariate more strongly associated with CD Anaerostipes, Methanobrevibacter,
≥3 months as defined by CAI for UC and analysis than gain of pathogenic ones) Christensenellaceae, Collinsella
CDAI for CD; 111 HCa
235 patients with IBD; diagnoses based 16S rRNA gene Ruminococcus gnavus (ileal Ruminococcus, Coprococcus, Blautia, 40

on clinical, radiological, endoscopic and
histological criteria; 38 HC
85 patients with IBD (43 UC, 42 CD);
sequencing of CD); Streptococcus anginosus Eubacterium, Dorea (IBD); Roseburia,
V3–V5 region; (IBD); Aggregatibacter segnis, Faecalibacterium, Dorea, Blautia
multivariate Actinobacillusb (in IBD flare vs (IBD flare); Anaerostipes (in IBD,
analysis remission); disease-​specific fungal particularly IBD flare and ileal CD)
microbiota dysbiosis
DNA shotgun Streptococcus salivarium Relative abundance of Roseburia 21

disease activity assessed by HBI for
CD and SCCAI for UC
123 patients with IBD (72 CD, 51 UC);
sequencing; neural (in patients with UC not achieving inulinivorans vs Burkholderiales at
network algorithms remission at week 14) baseline: Bifidobacterium longum,
Eggerthella, R. gnavus, R. inulinivorans,
Veillonella parvula (in patients with
CD achieving remission at week 14)
16S rRNA gene Streptococcus, Proteobacteria, Roseburia, Coprococcus, Clostridiales 71

diagnoses based on standard sequencing of Enterococcaceae (CD);

endoscopic, radiographic and V4 region; random Bacteroidia, Pseudomonadaceae
histological criteria; 73 HC forest models (UC)
447 newly diagnosed (treatment-​naive) 16S rRNA gene Pasteurellaceae, Veillonellaceae, Bacteroides, Clostridiales, 43

paediatric patients with CD; 221 HC sequencing of V4 Neisseriaceae, Fusobacteriaceae Faecalibacterium, Roseburia, Blautia,
region; multivariate and E. coli Ruminococcus, Lachnospiraceae
analysis
29 patients with CD receiving 16S rRNA gene Sulfate-​reducing Akkermansia, Barnesiella, 20

HSCT; diagnoses based on standard sequencing of Gammaproteobacteria Oscillibacter, Roseburia, Odoribacter

endoscopic, radiographic and V3–V4 region; and Deltaproteobacteria,
histological criteria random forest butyrate-​producing Clostridiales,
models Enterococcus, Megasphaera,
Campylobacter, Fusobacterium
300 patients with UC and 213 with CD 16S rRNA gene Firmicutes, Proteobacteria Dorea 158

assessed by HBI for CD and SCCAI for sequencing of

UC; 30 HC V3 region
62 individuals (11 healthy Lithuanian 16S rRNA gene Lachnospiraceae, Actinobacteria, Proteobacteria 101

dizygotic twin pairs, 7 healthy Lithuanian sequencing of Ruminococcaceae

monozygotic twin pairs, 8 German V4–V5 region
monozygotic twin pairs discordant
for UC and 10 German unrelated
HC); disease activity assessed by CAI,
endoscopic appearance and continuity
of inflammation, histology, and proven
exclusive involvement of the colon
CAI, colitis activity index; CD, Crohn’s disease; CDAI, Crohn’s disease activity index; HBI, Harvey–Bradshaw Index; HC, healthy controls; HSCT, haematopoietic stem
cell transplantation; IBD, inflammatory bowel disease; SCCAI, Simple Clinical Colitis Activity Index; UC, ulcerative colitis. aWithout a history of chronic disease.
b
Two members of the Pasteurellaceae family.

Dysbiosis
(Also known as pathobiome).
An altered microbial
community composition that
affects the host immune
response and leads to the
emergence and outbreak
of pathogens.
comprehensive view of functional dysbiosis and the
perturbations of metabolic pathways occurring in
IBD18,43,45–47. These studies showed that metabolic path-
ways involved in the synthesis of sulfur-​containing
amino acids, riboflavin metabolism, glutathione trans-
porters, oxidative stress and nutrient transport were all
upregulated18,43,45–47. One study with the ability to resolve
microbial communities down to the level of individ-
ual strains within a species revealed increased strain
diversity of pathobionts and reduced strain diversity
of beneficial microbes in stool samples from patients
with IBD or irritable bowel syndrome (IBS) com-
pared with healthy controls47. In-​depth analysis showed
that 219 taxa (including 152 species) were associated
with CD and 102 taxa (including 93 species) were asso-
ciated with UC47. CD was predominantly characterized
by a decrease in taxa belonging to Lachnospiraceae and
Ruminococcaceae and an increase in taxa belonging to
Enterobacteriaceae, whereas a decrease in taxa belong-
ing to Bacteroidaceae and an increase in taxa belonging
to Lachnospiraceae was observed for UC47. In concord-
ance with this heterogeneity, changes in only a few spe-
cies were shared across different IBD studies48, which
suggests that interindividual differences persist within
groups of patients with CD despite similar disease
phenotypes and disease courses.
One of the first pieces of clinical evidence for a patho-
genetic role of the intestinal microbiota in IBD origi-
nated from experiments showing that diversion of the
faecal stream from an inflamed segment of the small
intestine in patients with CD led to improvements in
disease symptoms49,50. Restoration of the faecal stream

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Table 2 | Functional risk profiling using shotgun metagenomics signatures
Study population
IBD
A subset of 4 patients with CD confirmed by
endoscopic, pathological or radiographic
findings and 7 HC subjected to shotgun
metagenomics to confirm 16S-​predicted
functional pathways generated from
121 patients with CD, 75 with UC and 27 HC
366 childrena with CD; disease activity
assessed by PCDAI
266 tissue samples obtained from
32 people (20 IBD, 12 HC); disease
activity assessed by CDAI or HBI for
CD and SCCAI for UC
1,792 participants (33 IBD, 412 IBS, 1,025
HC); diagnoses confirmed by endoscopic,
pathological or radiographic findings
155 individuals (68 CD, 53 UC, 34
non-​IBD controls); diagnoses confirmed by
endoscopic, pathological or radiographic
findings
Longitudinal sample of 1,638 stool samples Faecalibacterium prausnitzii and R. hominis; nicotinuric
from 132 patients with IBD; disease activity acid exclusively found (IBD)
assessed by HBI for CD or SCCAI for UC
T2DM
46 patients with T2DM, 442 with
prediabetes and 53 HC; assessed by
OGTT and a Finnish diabetes risk score
(FINDRISC)
53 patients with T2DM, 49 with impaired
glucose tolerance and 43 HC; assessed by
fasting blood glucose and HbA1c
1,179 patients from the LL-​DEEP study;
assessed by OGTT
77 patients with T2DM, 80 individuals with
prediabetes and 97 HC; assessed by GABA
degradation and pathway PWY-5022
activity
71 patients with T2DM and 74 HC
ABC, ATP-​binding cassette; CD, Crohn’s disease; CDAI, CD activity index; GABA, γ-​aminobutyric acid; HbA1c, glycated haemoglobin; HBI, Harvey–Bradshaw index;
HC, healthy controls; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; IQGAP1, RAS GTPase-​activating-​like protein IQGAP1; KEGG, Kyoto
Encyclopedia of Genes and Genomes; MYO1C, myosin IC; NR, not reported; OGTT, oral glucose tolerance test; PCDAI; paediatric CD activity index; SCCAI, simple
clinical colitis activity index; T2DM, type 2 diabetes mellitus; UC, ulcerative colitis. aAged under 22 years.
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Functional risk signature Ref.
Increased Decreased
Modules involved in glycolysis and carbohydrate Abundance of genes involved in lipid 45
transport and metabolism (nutrient uptake): metabolism and catabolism; global
metabolism of sulfur-​containing amino acids and decrease in nicotinamide, purine and
cysteine (compounds advantageous for oxidative pyrimidine nucleotide biosynthesis in IBD
stress), riboflavin metabolism and glutathione
transporters increased in IBD
Most predictive pathways: sulfur relay systems, Most predictive pathways: glycerophos- 46
galactose metabolism, biosynthesis of siderophores, pholipid metabolism, aminobenzoate
glycolipid metabolism, glutamine and/or glutamate degradation, sulfur relay systems and
metabolism, glutathione metabolism and biosynthesis selenocompound metabolism
of siderophores
Facultative anaerobe abundance (Streptococcus Blautia obeum, Alistipes putredinis (IBD) 77
salivarius, Streptococcus parasanguinis), Ruminococcus
gnavus (IBD); upregulation of genes involved in
oxidative stress (NADH oxidase and peroxiredoxin),
cysteine biosynthesis, acquisition of iron and 13 genes
involved in sugar utilization and transport
219 taxa (152 species) associated with CD; 102 taxa Taxa belonging to Lachnospiraceae and 133
(93 species) associated with UC; taxa belonging to Ruminococcaceae (CD); taxa belonging
Enterobacteriaceae (CD); perturbations in multiple to Bacteroidaceae (UC)
functional pathways, including pathways involved in
amino acid synthesis, vitamins, neurotransmitters and
short-​chain fatty acid synthesis (IBD); taxa belonging to
Lachnospiraceae (UC)
Unclassified Roseburia species (CD, UC); Bifidobacterium Roseburia hominis, Doreaformicigenerans 103
breve, Clostridium symbiosum uniquely enriched (UC); and Ruminococcus obeum strongly
12 species enriched, including R. gnavus, Escherichia coli enriched in controls without IBD
and Clostridium clostridioforme (CD)
E. coli, Ruminococcus torques, R. gnavus, 18
Clostridium hathewayi, Clostridium
bolteae; pantothenate and nicotinate
(vitamins B5 and B3) depleted (IBD)
118 species with significantly altered metagenomics NR 136
in two-​component systems: fructose and mannose
metabolism; pentose phosphate pathway;
branched-​chain amino acid synthesis and B group
vitamin (specifically vitamin B7) metabolism
Four members of Lactobacillus; metagenomic gene Five members of Clostridium 62
cluster model identified Roseburia and F. prausnitzii as
highly discriminant for T2DM; 7 of the T2DM-​enriched
KEGG orthologue markers; starch and glucose
metabolism; fructose and mannose metabolism; ABC
transporters for amino acids; ions and simple sugars;
cysteine and methionine metabolism
GABA degradation and pathway PWY-5022 activity NR 159
The most discriminatory metagenomic linkage group for Higher levels of four antimicrobial 61
separating treatment-​naive T2DM and normal glucose peptides in HC; IQGAP1 and
tolerance was Akkermansia muciniphila; F. prausnitzii unconventional MYO1C were uniquely
and E. coli were important in distinguishing prediabetes identified in the HC group; lower levels
from T2DM and HC; trimethylamine N-​oxide-​producing of proteases (trypsin and chymotrypsin
enzyme, dimethylaniline monooxygenase; higher and their precursors) and lipases
amylase 1 levels; antimicrobial cathepsin G
Bacteroides caccae, C. hathewayi, Clostridium ramosum, Clostridiales sp. SS3/4, Eubacterium 160
Clostridium symbiosum, Eggerthella lenta, E. coli; rectale, F. prausnitzii, Roseburia intestinalis,
membrane sugar transport; branched-​chain amino Roseburia inulinivorans, Haemophilus
acid transport; methane metabolism; xenobiotic parainfluenzae; bacterial chemotaxis,
degradation and metabolism; sulfate reduction flagellar assembly, butyrate biosynthesis
and metabolism of cofactors and vitamins
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and postoperative exposure of the neo-​terminal ileum to
the gut luminal contents induced inflammation, which
suggested that the intestinal microbiota triggers postop-
erative recurrence of CD49,50. Furthermore, the efficacy
of antibiotic treatment in subsets of patients with active
CD emphasizes the causal link between gut bacteria
and IBD51.
Mechanistic studies in mouse models of acute
and chronic intestinal inflammation provide further
evidence of a causal relationship between microbial
dysbiosis and IBD52,53. For example, FMT from patients
with IBD to germ-​free recipient mice was sufficient to
transmit the IBD phenotype20,54 and mice with a genetic
predisposition to IBD do not develop spontaneous
inflammation when kept under germ-​free conditions55.
Additionally, the transfer of dysbiotic microbial commu-
nities from genetically susceptible mice after the onset of
IBD is capable of transferring this disease phenotype to
germ-​free recipient mice56. The transfer of IBD micro-
biota into germ-​free, wild-​type mice induced an imbal-
ance in the intestinal T cell response leading to increased
numbers of intestinal T helper 17 (TH17) cells and TH2
cells and decreased numbers of RORγt+ (retinoid-​related
orphan receptor-​γ short isoform) regulatory T (Treg)
cells. The proportions of microbiota-​induced TH17
and RORγt+ Treg cells were predictive of human donor
disease status in the Rag1−/− mouse model of colitis (in
which colitis develops after transfer of naive T cells)
when these mice were transplanted with human IBD
microbiota57. Similarly, colonization of germ-​free mice
by Bacteroides fragilis, a human commensal bacterium
of the intestinal microbiota, induced the conversion of
CD4+ T cells into IL-10-​producing FOXP3+ Treg cells58,
which suggests the existence of microbiota-​driven
disease mechanisms in IBD.
Microbial dysbiosis in T2DM. Widely variable changes
in the abundance of several bacterial taxa have also
been described in T2DM (Tables 2,3). For instance,
an increased relative abundance of E. coli, Veillonella,
Blautia, Anaerostipes, Lactobacillus, Faecalibacterium
and Clostridiales (amongst others) has been reported
in patients with T2DM. Conversely, a reduced abun-
dance of Bacteroides, Bifidobacterium, Parabacteroides,
Oscillospira and the mucin-​degrading gut bacterium
Akkermansia muciniphila is associated with improved
metabolic health 19,59,60. A metagenomic and metap-
roteomic study published in 2019 analysed the gut
microbiota composition and function in faecal sam-
ples obtained from 254 Chinese individuals, including
77 treatment-​naive patients with T2DM, 80 people with
prediabetes and 97 control individuals with normal glu-
cose tolerance61. Patients with T2DM and individuals
with prediabetes had a lower abundance of bacterial spe-
cies belonging to Clostridiales and a higher abundance of
Megasphaera elsdenii compared to metabolically healthy
controls. Functional differences were observed in the
microbiomes of patients with T2DM and individuals
with prediabetes. The gut microbiota of individuals with
prediabetes showed enrichment of pathways involved in
sugar phosphotransferase systems, bacterial secretion
systems and ATP-​binding cassette (ABC) transporters of
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amino acids compared with that of control individuals.
These findings suggest that disease-​specific changes in
the gut microbiome can be detected in individuals with
prediabetes before they transition to T2DM. Differences
in bacterial species and metabolic gene cluster profiles
have been used to identify diabetes status in a set of indi-
viduals with either normal glucose tolerance or T2DM62.
However, confounding factors including geographic
location, ethnicity, health status and medication history
led to inconsistency in identifying microbial alterations
associated with T2DM60.
Several studies have provided evidence of a causal
link between specific members of the intestinal micro-
biota and the pathogenesis of T2DM. For example,
A. muciniphila is among the taxa shown to have a pro-
tective effect in metabolic disorders in both human
and mouse studies63–65. Interestingly, supplementation
with prebiotics normalized the abundance of A. mucin-
iphila and improved metabolic health in humans65.
Likewise, administration of A. muciniphila to mice
fed a high-​fat diet reversed their increased fat mass,
metabolic endotoxaemia, adipose tissue inflammation
and insulin resistance66. Despite its high oxygen sensi-
tivity and need for animal-​derived compounds in the
growth medium, A. muciniphila retains its protective
effects in mice when this organism is grown in a syn-
thetic medium compatible with human administration67,
which opens avenues for its development as a treatment
for human obesity and associated disorders. Further,
the butyrate-​producing bacterium Anaerobutyricum
soehngenii (previously designated Eubacterium hallii
strain L2-7) showed an increased abundance that cor-
related with improved peripheral insulin sensitivity in
recipients of FMT from lean donors29. Administration
of A. soehngenii to patients with T2DM improved their
peripheral insulin sensitivity after 4 weeks of treat-
ment, and these benefits were accompanied by an
altered microbiota composition and changes in bile
acid metabolism68.
Overlapping microbiome signatures. Curiously, spe-
cific bacterial taxa show similar alterations in both
IBD and T2DM, suggesting that common features of
immune-​mediated and metabolic disease lead to simi-
lar adaptations of the microbiota. Examples include the
decreased levels of Clostridium spp., Faecalibacterium,
Ruminococcus, Akkermansia, Collinsella and Roseburia,
and the increased representation of Enterobacteriaceae,
E. coli and Fusobacterium nucleatum spp., which
emphasize the challenges of defining disease-​specific
markers (Figs 1,2). As an example, the authors of a
study of 2,045 patients with IBD identified a signature
of eight taxa, including unknown members of the fam-
ily Christensenellaceae and the genus Fusobacterium,
that could discriminate between patients with CD and
healthy individuals17. However, an increased abundance
of Christensenellaceae is associated with both low BMI
and weight loss69, a catabolic condition that is frequently
observed in patients with IBD. Similarly, enrichment
of Fusobacterium is considered to be a marker of poor
prognosis in patients with metastatic colorectal cancer70.
Given that patients with IBD have an increased risk of
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developing colorectal cancer, this proposed microbiome
signature could be an epiphenomenon with no causal
link to the underlying disease mechanisms.
Additional meta-​omics approaches, including shot-
gun metagenomics and metabolomics, together with
the patient’s treatment history, demographics and
environmental data, have enabled detailed charac-
terization of the functional capacity of the gut micro­
biome. Findings from the second phase of the Human
Microbiome Project (HMP) immensely improved our
understanding of microorganism–metabolite interac-
tions in T2DM71 and IBD18. Integrative network analysis

Table 3 | Gut microbiome signatures associated with T2DM

Study population Methodology Risk signature Ref.
Increased Decreased
427 patients from the 16S rRNA gene sequencing of Escherichia coli, Veillonella, NR 93

CARE-​In-​DEEP Study, assessed V4 region; random forest Blautia, Anaerostipes

by OGTT models; gene expression analysis
145 women with normal, Shotgun metagenomics; random Lactobacillus gasseri, Desulfurispirillum indicum, Clostridium 62

impaired or diabetic glucose forest models Lactobacillus salivarius beijerinckii, Clostridium eklund,
control, assessed by HbA1c Clostridium botulinum, Pyramidobacter,
Clostridium thermocellum
800 patients with T2DM 16S rRNA gene sequencing Bacteroides thetaiotaomicron, Eubacterium rectale, Parabacteroides 90

of V3–V4 region; stochastic Alistipes putredinis distasonis, Roseburia inulinivorans,

gradient-​boosting regression Eubacterium eligens, Bacteroides vulgatus
60 individuals assessed by 16S rRNA gene sequencing of Bacteroidetes, Verrucomicrobia, Verrucomicrobia, Elusimicrobia, 95

fasting blood glucose levels
6,896 individuals with
V3–V4 region
16S rRNA gene sequencing
Proteobacteria, Elusimicrobia, Methanogenic archaeon
Acidobacteria, Deferribacteres,
Gemmatimonadetes,
Porphyromonadaceae,
Alistipes marseilloanorexic,
Bacillus sporothermodurans,
Staphylococcus, Prevotella
Actinobacteria, Fusobacterium, Akkermansia, Synergistes, 89

metabolic syndromea of V4 region; multivariate Streptococcus, Lactobacillus Methanobrevibacter, Oscillospira,

analysis Roseburia, Bifidobacterium
60 individuals assessed by 16S rRNA gene sequencing Faecalibacterium, Clostridiales, Bifidobacterium, Parabacteroides, 94

fasting blood glucose levels of V3–V4 region; random forest Dorea, Clostridiaceae, Oscillospira, Bacteroides
models Lachnospiraceae
1,832 patients included in 16S rRNA gene sequencing of Lactobacillaceae Mogibacteriaceae, Clostridiaceae, 92

3 cohort studies, assessed V1–V2 region; gradient-boosting Butyrivibrio, Roseburia, Megamonas,

by fasting blood glucose or algorithm Dorea
HbA1c levels
784 patients from the Inter99 Shotgun metagenomics Lactobacillus, E. coli Roseburia, Subdoligranulum, 59

study, assessed by ADA HbA1c Intestinibacter

291 patients from the Africa 16S rRNA gene sequencing Peptostreptococcus, Eubacterium, Collinsella, Adlercreutzia, Anaerostipes, 161

America Diabetes Mellitus of V4 region; gene expression Prevotella, Desulfovibrio Epulopiscium, Clostridium butyricum,
study, assessed by ADA OGTT analysis Ruminococcus, Pediococcus
1,976 patients in the KORA 16S rRNA gene sequencing E. coli Faecalibacterium prausnitzii, 19

cohort assessed by WHO of V3–V4 region; random forest Bifidobacterium longum, Intestinales
OGTT models bartlettii, Coprococcus, Eubacterium
rectale
436 patients from the Popgen 16S rRNA gene sequencing B. thetaiotaomicron Clostridium sensu stricto, 91

cohort and 844 patients of V1–V2 region; multivariate E. coli, Romboutsia, Barnesiella,
from the FoCus cohort with analysis Pseudoflavonifractor, Veillonella,
HOMA-​IR >5.0 Roseburia
977 individuals with 16S rRNA gene sequencing NR Christensenellaceae 69

overweightb or obesityb of V4 region

from the TwinUK cohort
690 patients from the CDC 16S rRNA gene sequencing Gemellaceae, Streptococcus, Parabacteroides, Clostridiaceae, 162

cohort and NYU study, of of V4 region Blautia Lachnospiraceae, Ruminococcaceae,

whom 246 had overweight, Clostridiales, Oscillospira
142 had obesity and 211 had
a healthy weightb
ADA, American Diabetic Association; CDC, Centers for Disease Control and Prevention; HbA1c, glycated haemoglobin; HOMA-​IR, homeostatic model assessment
for insulin resistance; NR, not reported; NYU, New York University; OGTT, oral glucose tolerance test; T2DM, type 2 diabetes mellitus; WHO, World Health
Association. aDefined as waist >90 cm (men) or >85 cm (women), fasting blood glucose ≥6.1 mmol/l (110 mg/dl) or a diagnosis of diabetes mellitus, blood
triglyceride levels ≥1.7 mmol/l (150 mg/dl), high-​density lipoprotein <1.04 mmol/l (40 mg/dl) and blood pressure ≥130/85 mmHg or a previous diagnosis of
hypertension. bDefined as follows: healthy weight, BMI 18.5–24.9 kg/m2; overweight, BMI 25.0–29.9 kg/m2; obesity, BMI ≥30 kg/m2.

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1 2 3 4 5
Enriched Enriched Enriched Enriched Decreased Enriched Decreased
Odoribacter Escherichia coli Escherichia coli Ruminococcus Ruminococcus Clostridiales Faecalibacterium
Oscillibacter Veillonella Clostridium gnavus Blautia Enterococcus Peptostreptococcaceae
Pseudoflavonifractor Blautia Romboutsia Streptococcus Coprococcus Megasphaera Anaerostipes
Barnesiella Anaerostipes Barnesiella anginosus Eubacterium Campylobacter Akkermansia
Decreased Pseudoflavonifractor Aggregatibacter Dorea Fusobacterium Methanobrevibacter
Faecalibacterium Veillonella segnis Roseburia Escherichia coli Collinsella
Butyricimonas Roseburia Actinobacillus Faecalibacterium Christensenellaceae
Decreased Pasteurellaceae Anaerostipes Barnesiella
1 Bacteroides Oscillibacter
Enriched Faecalibacterium Roseburia
Pasteurellaceae Bifidobacterium Odoribacter
Veillonellaceae Eubacterium
Neisseriaceae Coprococcus
6
Fusobacteriaceae
Escherichia coli Decreased
Bifidobacterium longum Christensenellaceae
Eggerthella 7,8
Ruminococcus gnavus 6
Roseburia inulinivorans 3 7 8 7 8
4 Enriched Enriched
Veillonella parvula
Streptocccus salivarius 1 5 Firmicutes Lactobacillus
9 11 Proteobacteria Escherichia coli
Decreased 10
Bacteroides 2 Decreased Decreased
Clostridiales Dorea Sulfurospirillum
12 Clostridium
Faecalibacterium
Roseburia inulinivorans Pyramidobacter
Blautia Roseburia
Ruminococcus Subdoligranulum
Lachnospiraceae Intestinibacter
Burkholderiales
Ruminococcus 9 12
Eggerthella
Enriched 10 11 Enriched
Bifidobacterium longum
Peptostreptococcus Enriched Decreased Enriched Decreased Peptostreptococcus
Ruminococcus gnavus
Eubacterium Proteobacteria Verrucomicrobia Actinobacteria Akkermansia Eubacterium
Veillonella parvula
Prevotella Acidobacteria Elusimicrobia Fusobacterium Synergistetes Prevotella
Lactobacillus salivarius
Desulfovibrio Deferribacteres Eubacterium Streptococcus Methanobrevibacter Desulfovibrio
Decreased Bacillus Parabacteroides Lactobacillus Oscillospira Decreased
Collinsella Prevotella Roseburia Faecalibacterium Roseburia Collinsella
Adlercreutzia Bacteroides Bacteroides vulgatus Clostridiales Bifidobacterium Adlercreutzia
Anaerostipes Thetaiotaomicron Dorea Parabacteroides Anaerostipes
Epulopiscium Alistipes Lachnospiraceae Mogibacteriaceae Epulopiscium
IBD Clostridium Gemmatimonadetes Lactobacillaceae Megamonas Clostridium butyricum
Ruminococcus Porphyromonadaceae Butyrivibrio Ruminococcus
T2DM Pediococcus Staphylococcus Dorea Pediococcus

Fig. 1 | Bacterial risk signatures in the gut microbiomes of patients with IBD or T2DM. Box colours reflect enriched
and decreased taxa in inflammatory bowel disease (IBD, blue) and type 2 diabetes mellitus (T2DM, purple) in diverse
populations across the globe.

of microbiome, metabolome and transcriptome data pharmacologic responses to a therapeutic interven-

sets from 132 individuals identified disease-​associated
network hubs that connect key bacterial taxa (namely
F. prausnitzii, unclassified Subdoligranulum, Alistipes,
E. coli and Roseburia) to certain metabolites (short-​chain
fatty acids, octanoyl carnitine and several lipids).
Interestingly, differences between study participants
with and without IBD were more apparent in the faecal
metabolome than in the faecal metagenome, faecal meta-
transcriptome or faecal proteome18. In the Integrated
Personal Omics Profiling Study (iPOP), plasma metab-
olites were strongly correlated with insulin resistance in
longitudinal samples from 106 individuals, which sug-
gests that interactions between the host metabolome and
the gut microbiome are perturbed in insulin-​resistant
individuals.
Biomarkers of gut dysbiosis
A National Institutes of Health working group has
defined a biomarker as “a characteristic that is objec-
tively measured and evaluated as an indicator of nor-
mal biological processes, pathological processes, or
tion”72. An ideal clinical biomarker should be rapid,
quantitative, objective, reproducible, non-​invasive and
exhibit high accuracy in predicting disease states across
multiple populations or ethnicities73. The identifica-
tion of microbiome biomarkers and their use for the
classification of disease entities require extensive use
of computational and statistical tools to identify net-
works of bacterial taxa that can accurately discriminate
between different disease phenotypes (such as healthy
individuals from patients with IBD, or individuals with
prediabetes from patients with T2DM) as well as dis-
tinguish closely related disease entities (such as IBD
and IBS). Profiles of microbial biomarkers also require
validation in large, independent, population-​b ased
cohorts to confirm their diagnostic or prognostic utility.
In the following sections, we review the progress made
towards the development of microbiome-​based bio-
markers for disease risk profiling. These biomarkers
increase in complexity from single indicator bacterial
strains or taxa through dysbiotic microbial communities
to multi-​omics-​based biomarkers (Box 1).

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Bacillus sporothermodurans
Staphylococcus
Lactobacillus gasseri
Eubacterium
Prevotella Pseudomonadaceae
Alistipes marseiloanorexi Actinobacillus
Lactobacillus salivarius Aggregatibacter segnis
Alistipes putredinis Dorea
Desulfovibrionales Megasphaera
Bacteroides thetaiotaomicron Enterococcus
Blautia Streptococcus salivarius
Parabacteroides
Ruminococcus Streptococcus anginosus
Megamonas Akkermansia Peptostreptococcus
Pediococcus Methanobrevibacter Fusobacterium
Aminicella Collinsella Campylobacter
Intestinibacter Roseburia Neisseriaceae
Clostridium butyricum
Clostridium botulinum Escherichia coli Ruminococcus gnavus
Clostridium beijerinckii Eggerthella
Subdoligranulum Veillonella
Oscillospira Clostridiales
Clostridium thermocellum Anaerostipes Oscillibacter
Epulopiscium Faecalibacterium Coprococcus
Eubacterium eligens Butyrivibrio
Eubacterium rectale Bifidobacterium longum Barnesiella
Desulfurispirillum Burkholderiales
Synergistetes
Pyramidobacter
Adlercreutzia
Bacteroides vulgatus

IBD T2DM Increased in T2DM Decreased in T2DM Increased in IBD Decreased in IBD

Fig. 2 | Overlapping global risk profiles in IBD and T2DM. Bacterial genera signatures associated with inflammatory
bowel disease (IBD) or type 2 diabetes mellitus (T2DM) and common to both diseases based on the results of 16S rRNA
gene sequencing microbiome association studies summarized in Tables 1–3.

Indicator strains and taxa. Many studies have inves-
tigated the utility of microbial alterations as disease
biomarkers, particularly in patients with CD or UC.
Initial efforts attempted to define single bacterial taxa
that could act as indicators of disease activity. For
instance, F. prausnitzii (a butyrate-​producing Firmicutes)
is depleted in patients with CD41. A reduced abundance
of this bacterium in ileal mucosa biopsy samples from
patients with CD strongly correlated with an increased
risk of endoscopic recurrence after ileal resection.
Conversely, an increased abundance of adherent inva-
sive E. coli correlated with ileal CD74. Most of these early
studies used 16S rRNA amplicon sequences to achieve
family-​level or genus-​level taxonomic classification;
only rarely were species-​level associations identified.
However, as most bacterial species consist of many
individual strains that can exhibit considerable genomic
and proteomic variation, strain diversity is of great func-
tional importance, particularly in determining patho-
genicity. For instance, subspecies of both R. gnavus and
E. coli have been associated with increased severity of
IBD75,76. Moreover, a particular subspecies of R. gnavus
that shows increased abundance in faecal samples from
IBD patients has been shown to contain strain-​specific
genes involved in improved bacterial colonization.
These genes involve functions such as oxidative stress
responses, bacterial adhesion, iron acquisition and
host mucus utilization77. Similarly, different strains of
B. fragilis showed functional divergence that resulted in
different levels of IgA induction in IBD-​related mouse
models78. These genetically distinct B. fragilis strains also
showed differential colitogenic and immunomodulatory
effects when inoculated into recipient mice78.
In a study that aimed to define key dysbiotic taxa for
use in monitoring of disease activity in patients with
IBD, the absolute abundance ratios of F. prausnitzii and
E. coli (also known as the F–E index) were computed
using quantitative PCR. Use of the F–E index improved
the discrimination of patients with UC and IBS from
those with CD and helped to discriminate colorectal
cancer from other gut disorders. However, the F–E index
could not discriminate between IBD subtypes79,80, which
suggests that single-​taxa indicators have limited util-
ity for classifying subtypes of a disease. Gut dysbiosis
indices have mostly included bacterial species only;
however, one study used the differential abundance
of two fungal phyla, Basidiomycota and Ascomycota,
which robustly discriminated samples originating from

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healthy individuals from samples obtained from patients
with IBD of different disease phenotypes40. In addition,
reduced fungal diversity and an increased abundance of
Candida taxa have been found in the gut microbiota
of paediatric patients with CD81. Interestingly, fungal and
bacterial loads differ markedly between healthy relatives
of patients with IBD and unrelated healthy controls as
well as between patients with different IBD subtypes.
These observations demonstrate that fungi as well
as bacteria contribute to IBD-​related gut dysbiosis82.
Dysbiosis scores. Several studies have evaluated the per-
formance of gut dysbiosis indices that describe increas-
ingly complex co-​o ccurrences of specific bacterial
abundances in disease classification. For instance,
one study found that the microbiota of patients with
IBD fluctuates more dramatically than that of healthy
individuals83. The authors of this study identified upper
and lower limits for the normal variation in the gut
microbiome of healthy individuals and termed the inter-
val between these limits the “healthy plane”83. The dis-
tance to the healthy plane varied over time in patients
with IBD but did not necessarily correlate with disease
activity83. In a large cohort of paediatric patients with
early-​onset CD (the RISK study), a microbiome dys-
biosis index (defined as the log of the total abundance
of organisms that are increased in individuals with
IBD divided by the total abundance of organisms that
are decreased in patients with IBD) strongly corre-
lated with disease severity and could be used to stratify
patients43. Nevertheless, this microbiome dysbiosis index
Box 1 | Gut microbiome biomarker discovery
The following processes are ordered by increasing data complexity.
Single taxa as indicator markers for disease
Expansion of pathobionts (for example, Escherichia coli, Ruminococcus gnavus).
Reduction of beneficial microbes (for example, Faecalibacterium prausnitzii).
Methodology:
• 16S rRNA gene sequencing: genus level
• Shotgun metagenomics: strain level
• Quantitative PCR for validation: co-​abundances of F. prausnitzii and E. coli (F–E index,
B/F ratio)
Microbiome dysbiotic index
A measure quantifying the deviation (shift) of microbiome composition from a healthy
microbiome baseline.
A ratio of the total abundance of organisms increased in inflammatory bowel disease
over the total abundance of organisms decreased in inflammatory bowel disease.
Methodology:
• 16S rRNA gene sequencing and shotgun metagenomics
• Dissimilarity metric (e.g. β-​diversity, Bray–Curtis) describing microbiome dissimilarity
between healthy and diseased states
Multi-​omics based biomarkers
The concurrent application of omics applications to achieve a global view of changes in
genetic, metabolic and biochemical processes in the gut microbiome. Using machine
learning, a subset of the features (candidate biomarkers) is generated and used to build
predictive models that can effectively discriminate between disease subtypes.
Methodology:
• Metagenomics, metatranscriptomics, metaproteomics, metabolomics
• Machine learning methods
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had a limited capacity to classify IBD disease subtypes,
and also showed overlap between patients with IBD and
healthy individuals43.
A study from the second phase of the integra-
tive HMP (iHMP) used a dysbiosis score based on
Bray–Curtis dissimilarities to non-​IBD metagenomes
to identify samples from patients with IBD that had
highly dysbiotic metagenomic microbial structures18.
In addition to the microbial response to inflammation,
this dysbiosis score encompassed other host-​related
factors (such as transcriptomic regulation) and bio-
chemical interactions between serum metabolites and
chemokines, which provided a comprehensive view of
systemic changes and microorganism–host interactions
in IBD18. Nevertheless, we emphasize that dysbiosis is
a poorly defined condition and thus different dysbio-
sis indices will differ according to the methodology
used to derive them, the disease entity investigated,
and their performance in different cohorts or groups of
individuals.
Large-​scale biomarker profiling. Several studies have
used machine learning algorithms to validate complex
microbiome signatures in cross-​sectional and longitu-
dinal patient cohorts. For example, a study published
in 2017 used 16S rRNA microbiota profiling to analyse
faecal samples from a large cohort of patients with IBD
and control individuals without IBD18. The researchers
used a sequence clustering algorithm to identify a CD-​
specific microbial signature based on the abundance of
eight bacterial taxa18. Additionally, another group showed
that characteristics of the gut microbiome composition
at baseline could predict the response of patients with
IBD to anti-​integrin therapy at 14 weeks after treatment
initiation. A microbiome signature generated by a deep
neural network had an area under the receiver operating
characteristic curve (AUC) of 0.87, compared to an AUC
of 0.62 for a model based on clinical covariates21.
Our group has also assessed the utility of microbiome
signatures as biomarkers of IBD and T2DM. In our first
study, we examined disease activity and response to ther-
apy in a unique cohort of 29 patients with CD who had
undergone autologous haematopoietic stem cell trans-
plantation. Integration of microbiome and metabolome
risk profiles from human donors and humanized mice
improved the performance of predictive models of dis-
ease outcomes from AUC 0.79 to 0.96 and identified a
network of disease-​associated bacteria and metabolites
involved in sulfur metabolism20. Although these findings
sound promising, it is important to acknowledge that
microbiome risk profiles are based on predictive models
derived from populations or groups of patients included
in prospective cohort studies, and hence could be more
accurate for groups of similar patients (either popula-
tions or cohorts) than they are for individuals. Therefore,
it is important to keep in mind that the predicted risks
might not translate directly to individual patients, pos-
sibly owing to limited generalizability of the risk profile
in heterogeneous settings.
Our second study investigated metabolic health and
the diurnal rhythmicity of gut microbiota in a German
population-​based cohort of 1,976 individuals. Faecal
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microbiota profiling identified a risk signature of
13 microbial taxa that showed disrupted diurnal rhyth-
micity in patients with T2DM. A predictive model based
on this arrhythmic risk signature successfully identified
individuals at risk of developing T2DM and had an AUC
of 0.78 when BMI was included in the model19. These
examples, among others, provide evidence for a role of
microbiome signatures in biomarker discovery for diag-
nostic and therapeutic purposes. However, it is impor-
tant to note that dysbiosis indices are not standalone
measurements and need to be integrated into additional
host-​derived and clinical data. Standardization and val-
idation of these indices require large-​scale studies that
include longitudinal assessment of potential biomark-
ers and consider possible confounding variables such
as diet, age, ethnicity, medical history and lastly time of
defecation, all of which have been proven to influence
microbiome alterations.
Inconsistency of disease biomarkers
Studies that compared populations from industrial-
ized countries with rural or non-​industrialized popu­
lations have identified dramatic differences in their
gut microbiome characteristics. A 2015 study com-
pared the faecal gut microbiota of individuals from
two non-​industrialized regions in Papua New Guinea
with those of individuals resident in the USA 84.
Interestingly, gut microbiome profiles in the Papua
New Guinea residents showed much higher microbial
diversity and lower interindividual variation compared
to those from US residents84. Many bacterial species
were common to both Papua New Guinea and US res-
idents but showed different abundance levels, which
were explained by decreased bacterial dispersal rates
in Western populations84. In another pioneering study
aimed at identifying a global core microbiome in healthy
populations, the number of core genera decreased from
17 to 14 when gut microbiome profiles from rural popu­
lations in Papua New Guinea, Peru and Tanzania were
compared with those from populations in industrialized
countries, including Flemish and Dutch cohorts as well
as UK and US populations85. This loss of resident micro-
bial species, termed the “disappearing microbes”86, might
help to explain the rising incidence of chronic diseases
in industrialized countries.
In a robust study conducted to validate the generaliz-
ability of several microbiota-​based classifying models of
metabolic health, the gut microbiota of 7,009 individuals
from 14 districts in a single Chinese province were char-
acterized to test the effect of geographic location on the
predictive power of the included models60. Interestingly,
host location showed the strongest association with
variations in gut microbiome60. Furthermore, in a large
longitudinal intercontinental study of 531 patients with
IBD from Ireland and Canada, geographic location
was the major determinant of microbiome variations,
although most (90.3%) of the variance in microbiome
composition remained unexplained87. The importance
of geographic factors and related environmental expo-
sures are also illustrated in migration studies, which
clearly demonstrate strong associations between migra-
tion and both microbiome functional diversity and
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strain diversity. In this context, 16S rRNA and deep
shotgun metagenomic sequencing has been performed
on stool samples collected from individuals living in
Thailand and the USA, including first-​generation and
second-​generation Thai immigrants to the USA before
and after immigration88. Intriguingly, US immigration
was associated with substantial alterations to the gut
microbiome, including loss of diversity, loss of bacterial
strains, functional loss of fibre degradation and a shift
from Prevotella to Bacteroides enterotypes. These per-
turbations were intensified by obesity and increased with
each successive generation88.
The changes reported in selected microbiome asso-
ciation studies from countries around the world reveal
important connections between geographical loca-
tion and gut microbial dysbiosis in individuals with
IBD and T2DM87,88. Owing to the ready availability of
16S rRNA gene-​sequencing data sets, many of these
cohort studies have used 16S microbial profiling despite
variability between studies in the sequenced 16S vari-
able regions or the sequencing platform used (Fig. 1).
In the IBD stool-​d erived microbiome, Firmicutes,
Proteobacteria and genera including Fusobacterium,
E. coli, R. gnavus and Streptococcus, consistently showed
an increased relative abundance that correlated with
IBD activity17,21,40,43. By contrast, Roseburia, Blautia
and Faecalibacterium consistently showed a decreased
relative abundance in these cohorts17,21,40,43. Most stud-
ies of T2DM showed a decrease in the relative abun-
dance of A. muciniphila, Clostridium, Roseburia and
Faecalibacterium89–92. However, substantial divergence
is evident in the disease-​associated microbiome profiles
of individuals from different geographical and ethnic
groups59,62,89,90,93–95 (Supplementary Tables 1,2). The rel-
evance of these differentially represented taxa to the
pathogenesis of IBD has been validated in several clini-
cal and translational gnotobiotic experiments as we have
already discussed29,58,63,67,80,96,97.
Collectively, these data indicate the necessity for
further global studies of human microbiota in differ-
ent geographic locations across continents to rule out
region-​associated confounding factors and define spe-
cific and individualized microbiome signatures. Studies
included within the iHMP are exploring the link between
gut microbiome alterations and the development of
IBD or T2DM in large population-​based cohorts18,71.
Nevertheless, to date, these studies have predominantly
focused on populations in industrialized countries
and most data have been obtained from the USA and
Europe, which represent ~17% of the world’s popula-
tion. A few national and international studies have been
initiated to characterize gut microbiome variations in
non-​European populations and ethnic groups, including
in Africa, Asia, South America and the Middle East98.
In all these studies, the authors have tried to identify
disease-​associated microbial risk profiles that contribute
to the development of either IBD or T2DM. However,
achieving a consensus on which disease-​related bac-
terial taxa have utility in the diagnosis of these two
diseases remains challenging. Despite the observed het-
erogeneity, a few bacterial taxa are indeed commonly
implicated across different studies (Fig. 1). For instance,
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Specificity
The proportion of individuals
who genuinely do not have the
disease or condition and
receive negative test results.
Sensitivity
The proportion of individuals
who genuinely have the
disease or condition and
receive positive test results.
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an increased abundance of Akkermansia, Eubacterium
rectale, Alistipes and F. prausnitzii is correlated with
improved metabolic health in multiple reports62,91,94,99,100.
Similarly, an overabundance of E. coli, Enterococcus,
Fusobacterium, R. gnavus and Streptococcus, and/or
a reduction in F. prausnitzii, Roseburia and members
of the Ruminococcus genus, are common features of
IBD-​ a ssociated dysbiosis 16–18,20,40,43,101. Intriguingly,
some taxa show similar trends in both IBD and T2DM.
For instance, an overabundance of Christensenellaceae
and E. coli has been linked to both CD-​associated and
T2DM-​associated dysbiosis17,19,94, which raises ques-
tions about the specificity of the available microbiome
signatures for discriminating between different dis-
ease entities. Looking at overlapping IBD-​associated
and T2DM-​associated core signatures showed similar
or opposite trends. The results of these 16S studies are
summarized in Tables 1–3. Additional detailed informa-
tion on these 16S studies is available in Supplementary
Tables 1 and 2. Bacterial signatures associated with each
disease entity are referred to by the highest reported tax-
onomic level included (bacterial phylum, family, genus
and species) (Fig. 2).
Metabolomics-​based biomarker discovery
In the search for disease biomarkers, metabolites serve
as the most proximal indicators of disease activity
and are strongly linked to the regulatory signals that
underlie and modulate disease mechanisms. In fact,
the metabolome and microbiome both fluctuate in
relation to endogenous and exogenous factors such as
diet, environment, ageing and overall health status102.
Numerous studies have reported substantial altera-
tions in the gut metabolite profiles of patients with
IBD18,20,103–105 or T2DM71,106–108. For instance, reduced lev-
els of medium-​chain fatty acids, such as pentanoate and
hexanoate109, and reduced levels of B vitamins110 have
been found in the faecal metabolome of patients with
IBD. Conversely, increased levels of amino acids, amines
and carnitines were reported in the faeces and sera of
adult and paediatric patients with IBD, respectively105,111.
The results of a landmark study showed that metabo-
lite profiling could discriminate patients with IBD
from healthy individuals112. This report was followed
by numerous others that consistently showed that the
metabolite phenotype of patients with IBD differs from
that of healthy individuals 103,104,113,114. Interestingly,
metabolite profiling could also discriminate between
different forms of IBD, such as CD and UC112,114, and
could further classify patients with CD as having either
ileal or colonic inflammation113.
Similarly, metabolite profiling of patients with
T2DM demonstrated altered activity of metabolic
pathways115,116. Serum levels of branched-​chain and aro-
matic amino acids, such as leucine, isoleucine, valine,
phenylalanine, tyrosine and tryptophan, were associated
with insulin resistance, obesity and the risk of develop-
ing T2DM in multiple reports117–119. Metabolite profiling
of patients with T2DM also revealed strong associations
between the levels of specific bacterial metabolites
and disease onset71,107,120,121. As an example, the trypto-
phan metabolism pathway includes several candidate
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metabolite biomarkers that have attracted research atten-
tion owing to their associations with the development of
inflammatory and metabolic disease in both human and
mouse studies122–125. Tryptophan is an essential amino
acid acquired from the diet and mainly absorbed in the
small intestine, although a small fraction is catabolized
to indole metabolites in the colon126. Tryptophan metab-
olism and downstream cellular signalling have been
extensively described in other reports126–128 and are not
discussed in this Review. One longitudinal cohort study
confirmed that high baseline fasting serum tryptophan
concentrations were associated with an increased risk of
developing T2DM in 213 Chinese individuals followed
up for 10 years (comprising 51 people who went on to
develop T2DM and 162 individuals who remained met-
abolically healthy)124. Moreover, the predictive power of
tryptophan levels as a biomarker was comparable to that
of five existing amino acids in discriminating between
individuals who did and did not develop T2DM124.
Of note, prior reports showed that serum levels of sev-
eral amino acids could identify patients with T2DM
from different populations with varying accuracy. For
example, phenylalanine and valine performed best in
American populations118, whereas tyrosine was most
accurate in South Asian populations129. These findings
point to the importance of identifying region-​specific
biomarkers in achieving optimal diagnostic accuracy.
Clinical role of microbiome biomarkers. A valuable bio-
marker must contribute additional classification power
to that derived from clinical information. As such, faecal
biomarkers are an obvious source of diagnostic mark-
ers of mucosal diseases given that the faecal stream
is in direct contact with the intestinal mucosa. Faecal
calprotectin, a granulocyte-​derived cytosolic protein
that can be detected in stool, is the most widely utilized
faecal biomarker for inflammatory disorders owing to
the strength of the correlation between inflammation
severity and faecal calprotectin levels130. Two reports
confirmed the ability of faecal calprotectin levels to
detect endoscopic inflammation, with a reported
sensitivity of 70–100% and specificity of 44–100%131,132.
The wide ranges of these values are explained by varia-
tions in the cut-​off thresholds applied in each study131,132.
Nevertheless, elevated levels of faecal calprotectin are
not specific for IBD but rather reflect inflammatory
conditions in the gut, which are also associated with
other intestinal and metabolic pathologies (including
IBS, gastrointestinal malignancies, obesity and T2DM).
For instance, shotgun metagenomic profiling of the gut
microbiota in stool samples from 1,792 individuals was
able to distinguish IBD from IBS, and machine learning
algorithms markedly improved the accuracy of these
prediction models (AUC 0.91) compared to that of faecal
calprotectin levels alone (AUC 0.80)133. Importantly, the
model with the highest prediction accuracy (AUC 0.93)
included faecal calprotectin levels as well as metagen-
omic profiling of the top 20 selected taxa133. These results
suggest that the integration of clinical and microbial
biomarkers improves diagnostic accuracy. Such an inte-
grated approach has been used to predict the response
to therapy in patients with IBD21. In this study, baseline
volume 19 | June 2022 | 393
Reviews

Identification of Validated
microbiome signatures microbiome signatures

Correlation Causation

Microbiome

function
Composition
D
Identification of environmental st isea Intervention studies

ns
io
factors at se Diet, FMT, post-biotics

ct
e

in xa
ra
Ta
Geography, diet

te
Multi-omics Microbial ecosystem therapeutics
Clinical relevance integration Identification and isolation of microbes
Cross-sectional and longitudinal Immune Geographical and metabolites, building functional
population and cohort studies status location consortia
ctors

Enviro
Microbe and host profiling ic D Gnotobiotic mouse models
net ha ieta
st fa

Microbiota (metagenome, metabolome) Ge eup

nme
k bi ry
ma ts
Ho

Host (phenotypes, genome,

Me sures

nt
exp
metabolome, immune response) Se e
Ag

dica
x

o
tion
High-throughput omics analyses Functional characterization
Biomarkers

Fig. 3 | Gut microbiome signature discovery in IBD and T2DM.
Investigation of the causative role of the gut microbiota in the pathogenesis
of complex diseases, such as inflammatory bowel disease (IBD) and type 2
diabetes mellitus (T2DM), requires large cross-​sectional or longitudinal
population-​b ased and cohort studies. Microbial, environmental
and host-​related factors need to be considered when establishing
correlations between disease entities and the structure or function of the
gut microbiome. Multi-​omics data are derived from high-​throughput
analysis of samples collected from the host (phenotype, genotype,
metabolome, transcriptome) as well as luminal or mucosa-​associated gut
microbiomes (composition, metabolic and genetic functional pathways).
Once candidate microbiome-​based biomarkers are identified using
complex computational and machine learning tools, functional validation
of candidate biomarkers is achieved through comprehensive in vivo, in vitro
and preclinical studies. Isolation and identification of candidate bacterial
taxa are performed for biobanking and for subsequent mechanistic studies
in gnotobiotic mice. Colonization of germ-​free mice with single bacterial
taxa or with synthetic minimal microbial consortia could give insight into
the causative role of specific microbes and the underlying microorganism–
host interactions. The clinical relevance of identified microbiome
signatures requires validation in faecal microbiota transfer (FMT) or dietary
intervention studies.

Diagnostic biomarkers
Characteristics that are directly
linked to the aetiology of the
disease such as elevated blood
glucose concentration (which is
diagnostic of type 2 diabetes
mellitus) and markers that
show a strong correlation with
inflammation in inflammatory
bowel disease (such as faecal
calprotectin levels).
clinical data (including serological, endoscopic and clin-
ical biomarkers) were insufficiently predictive of remis-
sion (AUC 0.62), whereas the addition of taxonomic
and metabolic profiles improved the diagnostic power
to AUC 0.72 and AUC 0.74, respectively21. Further, faecal
calprotectin levels alone were able to distinguish patients
with pouchitis from those without pouchitis (AUC
0.63)134. By contrast, a microbiome species model (with
or without faecal calprotectin level as an additional pre-
dictor) achieved an AUC of 0.78, confirming that micro-
bial profiling had a superior diagnostic performance to
faecal calprotectin levels alone in the identification of
pouchitis134.
Serological biomarkers of impaired glucose metab-
olism used in the diagnosis of T2DM include fasting
plasma glucose levels, 2 h plasma glucose levels after
a 75 g oral glucose challenge (the oral glucose tol-
erance test) and levels of glycated haemoglobin135.
Combinations of serological and microbiome biomark-
ers that predicted T2DM progression were identified in
a study that used data from two Swedish cohorts136.
In this study, multivariate analyses demonstrated a
strong correlation between the degree of insulin resist-
ance and microbiome variations136. Interestingly, the
use of a microbiome-​based machine learning model
to distinguish between individuals with the lowest
and the highest degree of insulin resistance in the val-
idation cohort yielded an AUC of 0.78, which suggests
that the gut microbiota is an important modifier of
T2DM progression136. In fact, although a broad range
of diagnostic biomarkers has been proposed for T2DM,
most of them fail to capture the complexity of this dis-
ease or to grasp both microbial and metabolic altera-
tions. In this regard, metabolite biomarkers have been
used in combination with established clinical risk factors
to substantially improve disease classification137,138.
Implications of microbiome signatures. Understanding of
the functional role and specificity of alterations in single
bacterial taxa (pathobionts)139,140 and/or complex micro-
bial communities (dysbiosis)141–143 is essential to resolve
the pathogenetic mechanisms of microorganism–host
interactions in IBD or T2DM (Fig. 3). In this context,
functional alterations of the gut microbiome potentially
represent the consequences of host adaptations.
A causal link of gut microbiota to multiple dis-
eases has been demonstrated in gnotobiotic mouse
experiments20,54,56,99,142,144–146. Germ-​free mouse models
can be selectively colonized with single bacterial strains,
minimal bacterial consortia or defined complex gut
microbial ecosystems derived from human stool or other
donor material to study their impact on host phenotype.

394 | June 2022 | volume 19 www.nature.com/nrgastro

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 Reviews

In IBD, single-​strain colonization of germ-​free mouse
models with a variety of commensal bacteria, including
E. coli, Enterococcus faecalis, Bacteroides vulgatus and
Bilophila wadsworthia has enabled us to understand
some of the underlying mechanisms of disease initiation
or protection147–149. The generation of well-​characterized
minimal bacterial consortia (such as SIHUMI150 and
Oligo MM 12,151) provided the means to investigate
more-​complex mechanisms of host–microorganism and
microorganism–microorganism crosstalk under both
physiological and pathological conditions. In addition,
germ-​free mice colonized with human faecal micro-
biota (also known as human microbiome-​associated
mice) have been used extensively to investigate the
underlying pathogenetic mechanisms of complex dis-
eases, including IBD, T2DM, obesity99,142, asthma and
malnutrition20,54,99,142,144–146,152–154.
Our research work has shown that gut bacteria
are required to drive inflammation in a mouse model
of colitis, and that these same bacteria are associated
with the risk of relapse in patients with CD20. Despite
the known limitations of incomplete human bacterial
transfer into germ-​free mice155, our model captured
key features of the disease-​associated microbiome sig-
natures. The transfer of gut microbiota into germ-​free
mice resulted in the successful transfer of several disease
states, thereby revealing shared functional metabolic
pathways implicated in inflammation20. These shared
microbiome signatures could lead to improvements in
the classification of disease activity. Similarly, previ-
ous work showing that glucose tolerance156 and insulin
resistance142,157 are influenced by the composition of the
gut microbiome has been verified by a series of FMT
studies in gnotobiotic mouse models142,143.
Conclusions
Over the past two decades, evidence from human and
mouse studies has revealed a fundamental role of the
intestinal microbiome in the pathogenesis of inflam-
matory and metabolic diseases such as IBD and T2DM.
Changes in the structure and function of the gut microbial
ecosystem, also termed dysbiotic microbiome signatures,
have been associated with disease activity, risk of relapse
or response to therapy in patients with these diseases.
Nevertheless, the complexity and multifactorial patho-
genesis of most of these diseases, as well as the existence
of a variety of confounding factors in human studies,
continue to present major challenges to the clinical
implementation of microbiome signatures in diagnosis,
predicting prognosis and treatment decision-​making.
In this context, the identification, isolation and cultiva-
tion of functionally relevant bacterial strains is needed,
along with isolation and identification of their metabo-
lites. As such, defining the functional capacity of a given
microbial signature can be achieved by metagenomics
and metabolomics approaches. Strain-​level shotgun
metagenomic sequencing can be used to identify the
microbial strains included in these signatures, including
keystone species that are present in low abundance yet
have an important role in disease development, and to
infer bacterial metabolic pathways, microbial interac-
tions and microbial metabolites that affect host physiol-
ogy. The establishment and use of well-​defined in vivo
gnotobiotic mouse models are expected to provide
fundamental information on the effect of microbiome
composition on host physiology and disease suscepti-
bility. To address the observed heterogeneity and inter-
individual variation in microbiome signatures, dense
microbiome sampling and disease modelling across
populations and ethnic groups should be performed to
improve the generalizability of predictive models. Thus,
the stratification of population and patient cohorts is
necessary to improve individual assessments of disease
risk. To ensure the reproducibility and between-​study
comparability of the findings of microbiome studies,
the specificity and sensitivity of microbiome signatures
must be assessed and validated in multicentre studies of
well-​characterized cohorts of patients.
Current microbiome research is moving beyond the
mere description of microbial community structures and
disease associations and progressing towards understand-
ing the causative roles of gut bacteria in the pathogenesis
of complex chronic disorders. These collective efforts are
expected to enhance microbiome modelling and advance
the development of microbiota-​based signatures and/or
risk profiles that can be utilized in clinical settings.
Published online 21 February 2022

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Acknowledgements
The authors’ research is funded by the Deutsche
Forschungsgemeinschaft (DFG, German Research Foundation) –
project number 395357507 (SFB 1371, Microbiome
Signatures) — and has received funding from the European
Union’s Horizon 2020 research and innovation programme
under grant agreement number 964590.
Author contributions
A.M. and D.H. contributed equally to all aspects of the article.
In addition, S.R. researched data for the article, contributed
substantially to discussion of its content, and reviewed and/or
edited the manuscript before submission.
Competing interests
The authors declare no competing interests.
Peer review information
Nature Reviews Gastroenterology & Hepatology thanks
Herbert Tilg, Chaysavanh Manichanh and the other, anony-
mous, reviewer(s) for their contribution to the peer review of
this work.
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Supplementary information
The online version contains supplementary material available
at https://doi.org/10.1038/s41575-022-00581-2.
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