Human Microbiota Research: Produced By: Nature, Nature Microbiology, With Support From

June 2019
www.nature.com/collections/microbiota-milestone
Human microbiota research
Produced by: Nature, Nature Microbiology, With support from:

Nature Reviews Microbiology and
Nature Medicine
M I L E S T O N E S I N H U M A N M I C R O B I O TA R E S E A R C H
A field is born (FOREWORD)
1944 Culturing anaerobes (MILESTONE 1)
1958 Faecal microbiota transplantation for Clostridioides difficile infection (MILESTONE 2)
1965 Gut microbiota transfer experiments in germ-free animals (MILESTONE 3)
1972 The microbiota influences metabolism of host-directed drugs (MILESTONE 4)
1981 Microbiota succession in early life (MILESTONE 5)
1996 Sequence-based identification of human associated microbiota (MILESTONE 6)
1998 Stability and individuality of adult microbiota (MILESTONE 7)
2003 Beyond bacteria: studies of other host-associated microorganisms (MILESTONE 8)
2004 Regulation of mucosal immunity by the microbiota (MILESTONE 9)
2005 The importance of adequately feeding your microbiota (MILESTONE 10)
2006 Transfer of host phenotypes through microbiota transplantation (MILESTONE 11)
2006 Impact of diet–microbiota interactions on human metabolism (MILESTONE 12)
2007 Mechanisms of colonization resistance (MILESTONE 13)
2007 Functional human microbiota analyses in vivo using ’omics technologies (MILESTONE 14)
2010 Antibiotic effects on microbiota composition and host health (MILESTONE 15)
2010 Bioinformatics tools enable the analysis of microbiome sequencing data (MILESTONE 16)
2010 Microbiome analyses in large human populations (MILESTONE 17)
2011 The microbiota–gut–brain axis (MILESTONE 18)
2012 Modern culturing efforts expand the culturable microbiota (MILESTONE 19)
2012 Global human microbiome (MILESTONE 20)
Microbially-produced short-chain fatty acids induce regulatory T cell production

2013 (MILESTONE 21)
2014 Production of antibiotics by the human microbiota (MILESTONE 22)
2015 Host-targeted drugs affect microbiota populations (MILESTONE 23)
2018 Human microbiota affects response to cancer therapy (MILESTONE 24)
Metagenome-assembled genomes provide unprecedented characterization of

2019 human-associated microbiota (MILESTONE 25)
S2 | JUNE 2019 www.nature.com/collections/microbiota-milestone

MILESTONES
Credit: Tetra Images / Alamy Stock Photo

F O R E WO R D Koch, Escherich, Kendall and a few
others, laid the foundations of how
A field is born
we understand host–microrganism
interactions. Pasteur developed the
germ theory of disease, but also
thought that non-pathogenic micro-
“I then most always saw, with of bacteria (although he called them organisms might have an important
great wonder, that in the said animalcules at the time) present in role in normal human physiology;
matter there were many very little his own mouth and that of others, Metchnikoff believed that microbiota
living animalcules, very prettily and subsequently also compared composition and its interactions
a-moving.” his own oral and faecal microbiota, with the host was essential for health;
— Antonie van Leeuwenhoek. determining that there are differences and Escherich was convinced that
between body sites as well as between understanding the endogenous flora
Despite being considered by many as health and disease. Some of the first was essential for understanding
a relatively modern field of research, direct observations of bacteria were the physiology of digestion and the
the first descriptions of human- of human-associated microbiota. pathology and therapy of intestinal
associated microbiota date back to Fast-forward a couple of cen- disease. Sound familiar? The themes
the 1670s–1680s, when Antonie van turies and, in 1853, Joseph Leidy we explore in these ‘Milestones in
Leeuwenhoek started using his newly published a book entitled A Flora human microbiota research’ largely
developed, handcrafted microscopes. and Fauna within Living Animals, brought to bear the hypotheses and
In a letter written to the Royal Society which some consider to be the early work of these microbiology
of London in 1683, he described origin of microbiota research. Then, giants, on the shoulders of which the
and illustrated five different kinds the work of Pasteur, Metchnikoff, field stands today.
NATURE MILESTONES | HUMAN MICROBIOTA RESEARCH JUNE 2019 | S3

MILESTONES
In 1890, Koch published his numbers of existing cells and how 25 milestones, we want to highlight
famous postulates, four criteria many could grow in the lab, what particular areas of research — both
the field [...] designed to establish a causative rela- became known as the ‘great plate established and burgeoning — that
took off in tionship between a microorganism count anomaly’. This key observa- have contributed to a better under-
earnest once and a disease, and during the first tion motivated the development standing of our microbial selves, as
methods half of the twentieth century, micro- of sequencing-based approaches well as methodological advances
biology became more focused on the to identify unculturable micro that have propelled the field for-
to culture identification of etiological agents of organisms, which were pioneered by ward. We also want to highlight
anaerobic disease. This was also likely due to Woese, Pace, Fox and others to study important but lesser known aspects
organisms were the fact that most bacterial pathogens environmental microorganisms and of the field, such as the fact that our
can grow in the presence of oxygen, subsequently adapted to the analysis microbiota is not just composed of
discovered in
whereas most members of the gut of human-associated communities, bacteria; that human-associated,
the 1940s and microbiota cannot and thus could providing an unprecedented view health-promoting microbial com-
1950s, when not be studied at the time. Alfred into their composition. A key step munities exist on all bodily surfaces,
members of Nissle, a German physician, isolated in popularising microbiota research, not only our gut; and, importantly,
the Escherichia coli Nissle 1917 strain which got it into the mainstream that to have a complete picture
the microbiota — which remains a commonly used news and made it a household con- of the functional capacity of our
were grown probiotic — in 1917. During World cept, was the finding by the Gordon microbiota and its roles in human
and studied in War I, when the first gut eukaryotic group, in 2006, that reconstituting health, we need to look beyond the
the laboratory microorganisms and bacteriophages mice with the microbial communities gut of white, Western populations.
were also described, Nissle noticed associated with a human disease state We thank the many researchers
that one soldier did not succumb to could transplant the phenotype to from all corners of the field who
dysentery and thought he might have the animals. This opened the door have advised on the different aspects
a protective microorganism in his to research trying to establish causal of this project, as well as those who
gut. He isolated the strain and later relationships between altered micro- have participated in the podcasts.
showed that it antagonized other bial communities and disease, which It is, of course, impossible to cover
pathogens, establishing the concept has become a cornerstone of the field. everything in a field as broad and
of colonization resistance, whereby Although the first use of faecal diverse as this one, but we hope to
human-associated microorganisms microbiota transplantation (FMT) in have captured the major steps for-
prevent the establishment of patho- Western medicine was published in ward. In our attempt to summarise
gens in the same niche. 1958 by Ben Eiseman and colleagues, almost 350 years of research, we will
Despite these early insights, the who successfully treated four people have unavoidably missed impor-
field only took off in earnest once suffering from pseudomembranous tant contributions and sincerely
methods to culture anaerobic organ- colitis (before Clostridioides difficile apologize for any unintended over-
isms were discovered in the 1940s was the known cause), FMT was sights. Although we have focussed
and 1950s, when members of the already used in ancient Chinese these milestones on the study of
microbiota were grown and studied medicine. Fourth-century Chinese human-associated microbiota, other
in the laboratory. This is where we medical literature mentions its use, vibrant research communities are
have chosen to start our timeline of by Ge Hong among others, to treat trying to understand plant- and
milestones, as increasing numbers food poisoning and severe diarrhoea. animal-associated, as well as envi-
of researchers became interested in In the sixteenth century, Li Shizhen ronmental, microbial communities.
understanding the composition and used oral administration of a ‘soup’ We hope that this journey through
function of the microbial communi- containing fresh, dry or fermented history will be inspirational and
ties that live on our different surfaces stool to treat abdominal diseases. we look forward to the exciting
and how they change throughout In seventeenth century Europe, the developments that are sure to come,
our lives. The realization that much Italian Fabrizio and the German ultimately aiming to harness our
of the normal physiology of conven- Paullini documented the use of FMT, understanding of microbial commu-
tional laboratory mice was missing and the American microbiologist nities to improve not only human
in germ-free mice, and could be Stan Falkow candidly recalled his health, but that of plants, animals
reconstituted through colonization role in preparing first-generation and ecosystems.
with bacteria obtained from faeces, poop pills to reconstitute the gut Nonia Pariente, Nature Microbiology
enabled the first in vivo experiments. communities of surgical patients a
Comparisons of germ-free and year before Eiseman and colleagues FURTHER READING Savage, D. C. Microbial
colonised animals in the 1960s led to published their work. biota of the human intestine: a tribute to some
pioneering scientists. Curr. Issues Intest. Microbiol.
observations that predicted much of We recognize that an enormous 2, 1–15 (2001) | Finegold, S. M. A century of
what has since been discovered using body of work precedes each anaerobes: a look backward and a call to arms.
Clin. Infect. Dis. 16, 453–457 (1993) | Falk, P. G.,
methodologies that enable more milestone that we have selected to Hooper, L. V., Midtvedt, T. & Gordon, J. I. Creating and
in-depth analyses. Despite advances highlight progress in this field. This maintaining the gastrointestinal ecosystem: what
in culturing microorganisms, it soon foreword aims to pay homage to we know and need to know from gnotobiology.
Microbiol. Mol. Biol. Rev. 62, 1157–1170 (1998) |
became apparent that there were some of these microbiota pioneers. Leidy, J. A Flora and Fauna Within Living Animals
gross discrepancies between the With this project, divided into (Smithsonian Institution, 1853).

MILESTONES
M I L E S TO N E 1 revealed that the majority of envi-

ronmental microbial biodiversity
Culturing anaerobes
remained uncultured, inspiring
a rebirth of culture techniques.
Recent culture-dependent efforts
to characterize the human micro-
Understanding the role of our micro- oxygen, yet the Hungate technique biota (see MILESTONE 19) utilised
biota in health and disease has long was still more efficient and enabled dilution culturing and culminated
been hampered by the strict growth the Hungate a wealth of anaerobes that had not in the development of culturomics;
requirements of many of its constitu- grown previously in surface cultures a high-throughput methodology
technique
ent members. Underpinning modern to be isolated for further study. using hundreds of different culture
day investigations into the vast com- […] enabled Alternative approaches used today conditions, prolonged incubations,
plexity and functions of the human a wealth of were also launched in the mid-late and matrix-assisted laser desorption/
microbiota are fundamental method- anaerobes that 1960s, namely the GasPak and the ionization–time of flight (MALDI–
ologies to culture anaerobic bacteria anaerobic glove-box. The former, a TOF) spectrometry, combined with
outside their natural environment.
had not grown self-contained combustion jar system, 16S ribosomal RNA gene sequencing
From the rudimentary oxy- previously quickly made surface culture of for the rapid identification of a great
gen-free culture methods in the era in surface anaerobic microorgansims accessible number of previously uncultured gut
of Pasteur, and subsequent advances cultures to be to more laboratories. The glove-box, a bacteria.
in surface culture in the early twen- sealed chamber with attached gloves, With a large proportion of the
tieth century, the mid-1900s saw a isolated for filled with anoxic gases, was also a human microbiota requiring oxy-
substantial expansion and refinement further study. popular choice, simplifying equip- gen-free growth conditions, early
of anaerobic culture techniques, ment and procedures for oxygen-free breakthroughs in anaerobic culture
largely due to the pioneering work of culture. were crucial in enabling more of
Robert. E. Hungate. In a 1944 study of As well as apparatus to create an our microbiota to be isolated and
cellulose-degrading microorganisms oxygen-free environment, culturing classified, and for their metabolism,
in the bovine rumen, his revolution- anaerobes requires appropriate distribution and roles within the
ary roll-tube approach enabled the media, which must have a low microbiota to be studied. Initial
successful culture of Clostridium cello- oxidation-reduction potential, as methodologies paved the way for
bioparus and, in 1950, he published a well as the substrates obtained by higher-throughput technologies
complete description of his technique. microorganisms in their natural that provide vital insights about the
The protocol used rubber-stoppered habitat. Many researchers working on functions of the bacteria inhabiting
tubes of boiled culture medium with Gas impermeable Bacteroides species were instrumental the human body, and their effects
stopper
cellulose agar, through which anoxic in determining the requirements of on the human host. Now, with our
gas was bubbled to remove any specific anaerobic microorganisms, understanding of the importance of
remaining oxygen. Firstly, passing this and a recent breakthrough in media the gut microbiota in human health
gas through a column of hot, reduced composition (the inclusion of antioxi- advancing by the day, we are even
copper wire excluded any oxygen dants) has since permitted the aerobic more indebted to these early research-
from the gas itself, and the subsequent growth of anaerobic bacteria. ers and their innovations enabling the
addition of a reducing agent to the Moving into the twenty-first culture of anaerobes.
medium removed residual traces of century, the advent of metagenomics Hannah Clark, Nature Protocols
oxygen. Rolling tubes under cold
water produced a thin layer of solid H2 + CO2 ORIGINAL ARTICLE Hungate, R. E. Studies on cellulose fermentation: I. The culture and physiology of an
agarose medium, and for the first (80 : 20)
anaerobic cellulose-digesting bacterium. J. Bacteriol. 48, 499–513 (1944).
FURTHER READING Hall, I. C. Practical methods in the purification of obligate anaerobes. J. Infect. Dis. 27,
time, anaerobiosis was maintained 576–590 (1920) | Hall, I. C. Differentiation and identification of the sporulating anaerobes. J. Infect. Dis. 30,
throughout manipulations using 445–504 (1922) | Hungate, R. E. The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14, 1–49 (1950) |
a constant flow of anoxic gas. The Bryant, M. P. & Doetsch, R. N. Factors necessary for the growth of Bacteroides succinogenes in the volatile acid
fraction of rumen fluid. Science 120, 944–945 (1954) | Moore W. E. C. Techniques for routine culture of fastidious
method, now known as ‘the Hungate Agar anaerobes. Intern. J. Syst. Bacteriol. 16, 173–190 (1966) | Brewer, J. H. & Allgeier, D. L. Safe self-contained carbon
technique’, is still in use to this day. dioxide-hydrogen anaerobic system. Appl. Microbiol. 14, 985–988 (1966) | Spears R. W. & Freter, R. Improved
isolation of anaerobic bacteria from the mouse cecum by maintaining continuous strict anaerobiosis. Proc. Soc.
Several modifications later Exp. Biol. Med. 124, 903–909 (1967) | Drasar, B. S. Cultivation of anaerobic intestinal bacteria. J. Pathol. Bacteriol. 94,
emerged, such as the VPI (Virginia
Credit: S. Fenwick / Springer Nature Limited
417–427 (1967) | Savage, D. C., Dubos, R. & Schaedler, R. W. The gastrointestinal epithelium and its autochthonous
Polytech Institute) method for larger- bacterial flora. J. Exp. Med. 127, 67–76 (1968) | Aranki, A. et al. Isolation of anaerobic bacteria from human gingiva
and mouse cecum by means of a simplified glove box procedure. Appl. Microbiol. 17, 568–576 (1969) | Hungate R. E.
scale culture introduced by Moore in Colony
Chapter IV: A roll tube method for cultivation of strict anaerobes. Method. Microbiol. 3, 117–132 (1969) | Sutter, V. L.
1966, using prereduced medium and & Finegold, S. M. Antibiotic disc susceptibility tests for rapid presumptive identification of gram-negative
prehardened roll tubes. Hungate also anaerobic bacteria. Appl. Microbiol. 21, 13–20 (1970) | Sonnenwirth, A. C. Evolution of anaerobic methodology.
Am. J. Clin. Nutr. 25, 1295–1298 (1972) | Holdeman, L. V. & Moore, W. E. C. Roll-tube techniques for anaerobic
made adaptations to culture meth- bacteria. Am. J. Clin. Nutr. 25, 1314–1317 (1972) | Salyers, A. A. Energy sources of major intestinal fermentative
anogens, the strictest of anaerobes, anaerobes. Am. J. Clin. Nutr. 32, 158–163 (1979) | Goodman, A. L. et al. Extensive personal human gut microbiota
culture collections characterized and manipulated in gnotobiotic mice. Proc. Natl Acad. Sci. USA 108, 6252–6257
reported in 1969. Others, such as (2011) | Lagier, J. C. et al. Microbial culturomics: paradigm shift in the human gut microbiome study. Clin. Microbiol.
Spears and Freter in 1967, similarly Infect. 18, 1185–1193 (2012) | Dione, N. et al. A quasi-universal medium to break the aerobic/anaerobic bacterial
recognised the importance of con- culture dichotomy in clinical microbiology. Clin. Microbiol. Infect. 22, 53–58 (2016) | Lagier, J. C. et al. Culturing the
human microbiota and culturomics. Nat. Rev. Micro. 16, 540–550 (2018).
tinuously avoiding any exposure to

MILESTONES
M I L E S TO N E 2 community structure in the colon.

Currently, a research consortium
Faecal microbiota transplantation is recruiting patients to a clinical

trial examining whether FMT is
safe, and can prevent recurrent
for Clostridioides difficile infection C. difficile-associated disease. FMT

could inhibit C. difficile by multiple
mechanisms, such as suppression by
antimicrobial peptides, inhibition
of spore germination and vegetative
growth, competition for nutrients,
and activation of colonization
resistance (MILESTONE 13).
To reduce costs and increase
Credit: Science Photo Library / Alamy Stock Photo
patient and clinician convenience,

oral delivery of faecal microbiota has
been tested. In 2017, a randomized
clinical trial showed that FMT deliv-
ery by oral capsule was non-inferior
to delivery via colonoscopy, suggest-
ing oral capsules could be an effective
treatment approach for recurrent
C. difficile infection.
FMT has also been investigated
for non-C. difficile indications, such
as ulcerative and drug-induced
In 1958, Eiseman et al. reported the and colleagues also noted this link colitis, and has shown some promise.
successful treatment of pseudomem- to prior broad-spectrum antibiotic FMT from lean donors has also been
branous enterocolitis using a faecal treatment in their patients and spec- shown to increase insulin sensitivity
enema. Since then, faecal microbiota ulated that disruption of the ‘healthy in men with metabolic syndrome.
transplantation (FMT) has become gut flora’ underlies infection. Thus, since the pioneering report
widely accepted as a successful rescue The intention of performing FMT from Eiseman and colleagues, FMT
treatment for recurrent Clostridioides is restoration of the normal function has become an effective therapy for
difficile infection. FMT is also being of the gut microbiota. In Eiseman recurrent C. difficile infection and
investigated for other indications. and colleagues’ report, culture shows promise for treating other
Eiseman and colleagues presented of stool samples obtained during diseases.
the cases of four patients with infection showed the presence of Louise Stone, Nature Reviews Urology
pseudomembranous enterocolitis. Staphylococcus aureus, which, at the
They used enemas with faeces from time, was considered to be a pos- ORIGINAL ARTICLE Eiseman B. et al. Fecal
enema as an adjunct in the treatment of
healthy donors after other therapy sible cause of pseudomembranous pseudomembranous enterocolitis. Surgery 44,
options failed. The patients had a enterocolitis. S. aureus disappeared 854–859 (1958).
FURTHER READING Khoruts A. & Sadowsky M. J.
rapid recovery from their symptoms. from the stool sample cultures Understanding the mechanisms of faecal
FMT is now C. difficile infection can cause after administration of a faecal microbiota transplantation. Nat. Rev. Gastro. Hep.
an effective debilitating diarrhoeal symptoms enema in association with clinical 13, 508–516 (2016) | Pamer, E. G. Fecal microbiota
transplantation: effectiveness, complexities, and
when the bacterial spores germinate improvement. The authors suggested
treatment lingering concerns. Mucosal Immunol. 7, 210–2014
into vegetative cells that produce that normal colonic non-patho- (2014) | Schwan A. et al. Relapsing Clostridium
option for enterotoxins, resulting in colonic gens displaced the colitis-causing difficile enterocolitis cured by rectal infusion of
homologous faeces. Lancet 322, 845 (1983) |
recurrent inflammation and the formation pathogen, which, decades later, was Gorbach S. L., Chang T. W. & Goldin B. Successful
C. difficile of ‘pseudomembranes’ consisting demonstrated to be C. difficile. treatment of relapsing Clostridium difficile colitis
with Lactobacillus GG. Lancet 330, 1519 (1987) |
of inflammatory cells, dead cells Since Eiseman and co-workers’
infection and and debris. Over the past couple of publication, others have reported
Tvede M, Rask-Madsen J. Bacteriotherapy for
Clostridium difficile diarrhoea. Lancet 1, 1156–
is believed decades, C. difficile infection has success using faecal enemas, 1160 (1989) | US National Library of Medicine.
Microbial Restoration for Individuals With One or
to normalize increased in incidence, morbidity Lactobacillus rhamnosus GG (a pro- More Recurrences of Clostridium Difficile
the microbial and mortality, and has become biotic) and bacteriotherapy (using a Associated Disease (CDAD) https://ClinicalTrials.
known as a ‘superbug’. Despite anti- mixture of facultatively aerobic and gov/show/NCT03548051 (2018) | Kao, D. et al.
diversity and biotics being the standard treatment anaerobic bacteria) to treat relapsing
Effect of oral capsule- vs. colonoscopy-delivered
fecal microbiota transplantation on recurrent
community for C. difficile infection, they also C. difficile enterocolitis. Clostridium difficile infection: a randomized
clinical trial. JAMA 318, 1985–1993 (2017) | Vrieze, A.
structure in often are the cause of infection owing FMT is now an effective treat- et al. Transfer of intestinal microbiota from lean
to their suppressive effects on native ment option for recurrent C. difficile
the colon donors increases insulin sensitivity in individuals
gut microbiota and subsequent infection and is believed to nor- with metabolic syndrome. Gastroenterology 143,
913–916 (2012).
overgrowth of C. difficile. Eiseman malize the microbial diversity and

MILESTONES
between different species of the

gut microbiota. For example, one
strain of Lactobacillaceae that could
7α-dehydroxylate bile acids in vitro
did not have the same catabolic
activity when transferred to germ-free
rats until additional bacterial strains
were introduced. Another study in
germ-free rats also showed the met-
abolic capacity of the gut microbiota,
specifically the reduction of bilirubin
to urobilins, which had been assumed
by some to be produced by the liver.
These initial studies laid the ground
for detailed work that explored the
Laboratory mice housed under germ-free conditions. Photograph links between microbial and host
courtesy of Taren M. Thron, California Institute of Technology. metabolism (MILESTONE 12).
In addition to exploring the met-
M I L E S TO N E 3 abolic capacity of the gut microbiota,
GF animals were also essential for
Gut microbiota transfer elucidating the close links between

bacteria and the host that determine
tissue homeostasis and immune
experiments in germ-free animals system development. One striking
example is determination of the role
of segmented filamentous bacteria
Germ-free animals are raised under cultures of several anaerobic isolates. (SFB), which had previously been
sterile conditions to prevent coloni- After one week, the numbers and shown to closely interact with the
zation with bacteria and other micro- localization of these bacterial strains In 1965, intestinal epithelium. Experiments
organisms. These animals (mostly in the gastrointestinal tract were Schaedler and that led to monoassociation of germ-
mice and rats, but also guinea pigs comparable to those observed in free mice with SFB showed that
and chicks) have a life span similar to the NCS mice and remained stable
colleagues these bacteria are key determinants
that of conventional, normally col- for several months, confirming the introduced of intestinal lymphocyte numbers
onized animals, although they seem feasibility of microbiota transfer a new use and phenotype in mice. Subsequent
to have a slower or impaired growth experiments and their usefulness for for germ- studies have identified many links
as well as some anatomical and studying bacterial gut colonization. between specific microbial taxa
physiological differences (such as an Importantly, transfer of a Bacteroides free animals: and/or molecules and host immune
enlarged cecum). By the 1960s, germ- strain partially reduced the cecum the transfer function (MILESTONE 9).
free animals were a well-established enlargement typical of germ-free of bacterial Germ-free animals have been,
tool in nutritional studies aiming to mice, and the offspring of germ-free and still are, indispensable tools for
cultures to
understand the contribution of the mice that had been colonized with studying functional relationships
intestinal microbiota to host dietary a mixture of strains inherited those germ-free between the microbiota and the
requirements (for example, with strains and subsequently showed mice […] have host — although, as always with
regard to the synthesis of vitamins). normal cecum size and structure. been essential animal studies, the comparability and
In 1965, Schaedler and colleagues These results directly showed the applicability of the results to humans
introduced a new use for GF animals: important and profound effect of the
to study the need to be verified. Nevertheless, the
the transfer of bacterial cultures to gut microbiota on host development effects of the early studies using these models have
germ-free mice. Such transfer experi- and physiology. gut microbiota inspired several avenues of micro-
ments have been essential in studying This landmark study paved the on the host biota research and highlighted the
the effects of the gut microbiota on way for further research on the important effects the gut microbiota
the host ever since. In their pivotal effects of the gut microbiota on ever since have on their host.
study, Schaedler and colleagues the host and on the interactions Lucia Brunello,
reported the results of feeding Nature Reviews Disease Primers
bacterial cultures isolated from the
ORIGINAL ARTICLES Schaedler, R.W., Dubos, R. & Costello, R. bacteria are indigenous intestinal bacteria that activate
gut of Nelson–Collins–Swiss (NCS)
Association of germfree mice with bacteria isolated from normal intraepithelial lymphocytes and induce MHC class II molecules and
mice (a colony of albino mice that mice. J. Exp. Med. 122, 77–82 (1965) | Gustafsson, B.E., Midtvedt, T. & fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in
are free of ordinary mouse pathogens Norman. A. Metabolism of cholic acid in germfree animals after the the ex-germ-free mouse. Microbiol. Immunol. 39, 555–562 (1995).
establishment in the intestinal tract of deconjugating and FURTHER READING Johansson, K.R. & Sarles, W.B. Some
as well as intestinal Escherichia coli 7α-dehydroxylating bacteria. Acta Pathol. Microbiol. Scand. 72, considerations of the biological importance of intestinal
and Proteus spp.) to germ-free mice. 433–443 (1968) | Gustafsson, B.E. & Sewander Lanke, L. Bilirubin and microörganisms. Bacteriol. Rev. 13, 25–45 (1949) | Sommer F. &
The germ-free mice were fed food urobilins in germfree, ex-germfree and conventional rats. J. Ex. Med. Bäckhed, F. The gut microbiota — masters of host development and
112, 975–981 (1960) | Umesaki, Y. et al. Segmented filamentous physiology. Nat. Rev. Microbiol. 11, 227–238 (2013).
inoculated with individual bacterial

MILESTONES
Credit: G. Marshall / Springer Nature Limited

M I L E S TO N E 4
The microbiota influences metabolism of

host-directed drugs
Peppercorn and Goldman demonstrated that microbiota in drug transformations. An increasing
the anti-inflammatory drug, salicylazosulfapyri- number of studies have confirmed the role of
dine, could be degraded in conventional rats and the microbiota, not limited to the gut, in drug
when cultured with human gut bacteria, but not metabolism and highlighted the implications for
in germ-free rats, indicating a role for the gut drug inactivation, efficacy and toxicity.
ORIGINAL ARTICLE Peppercorn, M. A. & Goldman, P. The role of (2013) | Liang, X. et al. Bidirectional interactions between
intestinal bacteria in the metabolism of salicylazosulfapyridine. indomethacin and the murine intestinal microbiota. eLife 4, e08973
J. Pharmacol. Exp. Ther. 181, 555–562 (1972). (2015) | Klatt, N. R. et al. Vaginal bacteria modify HIV tenofovir
FURTHER READING Clayton, T. A. et al. Pharmacometabonomic microbicide efficacy in African women. Science 356, 938–945 (2017) |
identification of a significant host-microbiome metabolic Zimmermann, M., Zimmermann-Kogadeeva, M., Wegmann, R. &
interaction affecting human drug metabolism. Proc. Natl Acad. Sci. Goodman, A. L. Separating host and microbiome contributions to
USA 106, 14728–14733 (2009) | Lindenbaum, J., Rund, D. G., drug pharmacokinetics and toxicity. Science 363, eaat9931 (2019) |
Butler, V. P. J., Tse-Eng, D. & Saha, J. R. Inactivation of digoxin by the Spanogiannopoulos, P., Bess, E. N., Carmody, R. N. & Turnbaugh, P. J.
gut flora: reversal by antibiotic therapy. N. Eng. J. Med. 305, 789–794 The microbial pharmacists within us: a metagenomic view of
(2010) | Wallace, B. D. et al. Alleviating cancer drug toxicity by xenobiotic metabolism. Nat. Rev. Microbiol. 14, 273–287 (2016) |
inhibiting a bacterial enzyme. Science 330, 831–835 (2010) | Haiser, Koppel, N., Maini Rekdal, V. & Balskus, E. P. Chemical
H. J. et al. Predicting and manipulating cardiac drug inactivation by transformation of xenobiotics by the human gut microbiota. Science
the human gut bacterium Eggerthella lenta. Science 341, 295–298 356, eaag2770 (2017).
M I L E S TO N E 5
Microbiota succession in early life

Early life experiences have complex in France that were either bottle-fed carbohydrate metabolism and the
and long-lasting effects that can or breastfed; and in the third study, biosynthesis of amino acids and
reach into adulthood — the same faecal bacterial communities from vitamins. By 2–3 years of age, a stable
can be said of the acquisition and breastfed infants, weaned children The infant gut microbiota develops that resembles
succession of our microbiota during and adults born in urban England microbiota that of the adults in the infant’s
the first years of life. The culmination and rural Nigeria, were compared. undergoes community (see MILESTONE 7).
of years of investigation from many These studies provided quantitative When colonization first occurs
laboratories has led to an in-depth measurements of specific bacterial
a period is an open question; however, most
characterization of postnatal taxa in early life, giving insight into of massive scientists think that the foetus
microbial acquisition and maturation the pioneer species that colonize the change in the develops in a sterile environment and
during the first years of life, and infant gut. This paved the way for first years that we acquire the bulk of our initial
has led to the realisation that this future high-resolution studies of microbiota during and immediately
represents a crucial window in our microbial succession in infants. of life after birth. Recently, a few studies
long-term development. With the advent of ‘omics’ have found traces of bacterial DNA
Early studies, dating as far back technologies in the following in the placenta, in the amniotic fluid
as 1900, described various aspects decades, our understanding of when that surrounds the foetus and in the
of bacterial succession in infants, the majority of our microbiota meconium — suggesting prenatal
but in 1981, three studies were are acquired, and of what species colonization. However, many
reported that set out to quantitatively are there, has heightened and the scientists think these findings could
characterize early acquisition of importance of host–microbiota– be the result of contamination and
gut commensals and to study how environment interactions during early the debate is ongoing. Regardless of
feeding shapes our initial microbiota. life has become realised. The infant possible exposure to microorganisms
In one study, development of the gut microbiota undergoes a period in utero, the foetus is exposed to
bacterial community was investigated of massive change in the first years of microbial molecules that cross the
in infants in Sheffield, England, by life. The initial microbiota adapts over placenta from the mother.
culturing specimens taken from the time and is shaped by the availability The first major exposure to
meconium (a baby’s first faeces), of different nutrients. As the infant microorganisms happens during
faeces, mouth and umbilicus in the consumes increasingly more complex delivery, and is highly dependent on
first six days of life. In another study, dietary substrates, there are shifts the mode of delivery. The microbiota
the faecal bacterial community was in composition and an enrichment of neonates that are born vaginally
compared between infant cohorts of bacterial functions related to are enriched in bacteria that resemble

Credit: S. Bradbrook / Springer Nature Limited
MILESTONES
The environment and people that

surround an infant are also a source
of microorganisms that can colonize
various body sites. Genetically
unrelated parents and even pets
share a high proportion of their
microbiota with infants. Genetics
also has a role in determining our
microbiota make-up, as evidenced by
associations between the heritability
of specific taxa and host genes.
The use of antimicrobials,
which is essential for preserving
life when infants acquire a serious
bacterial infection, can impact the
ecological succession of the infant
microbiota. Antibiotics can impair
the diversity and stability of the
developing microbiota in infants, with
abundances of specific taxa remaining
the maternal vaginal microbiota life. Breastmilk contains a complex reduced for years after treatment. The
(for example, Lactobacillus species), community of bacteria that may help impact of antibiotics on the infant
whereas neonates delivered by cae- seed the infant gut microbiota, and in microbiota could have long-lasting
sarean (C-) section lack these species breastfed infants the gut microbiota is health implications and their use
and are instead enriched in skin dominated by species that metabolise in early life has been linked to an
commensals such as Staphylococcus, human milk oligosaccharides. increased risk of several diseases,
Streptococcus and Propionibacterium Overall, diet has been found to including asthma, inflammatory
species. Over time, these differences be a major determinant of the bowel disease and allergies (see
gradually reduce between vaginally infant gut microbiota. Studies of MILESTONE 9). More research is
and C-section-born infants; however, malnourished infants have shown required to uncover the underlying
in one study, bacteria associated with that maturation of the gut microbiota mechanisms; however, what is clear
C-section remained associated does not occur in a similar manner is that the microbiota has a vital role
with C-section-delivered infants up to healthy infants, even after dietary in immune, endocrine, metabolic
to two years of age, showing that intervention, and it has been and a variety of other developmental
birth mode could have long-term proposed that an ‘undernourished’ pathways in infants, and without it we
impacts on the microbiota. microbiome in infancy can would not be here today.
Postnatal factors further perpetuate growth impairments Ashley York,
configure the microbiota in early later in life. Nature Reviews Microbiology
ORIGINAL ARTICLES Rotimi, V. O. & Duerden, B. I. The Diversity of Bifidobacteria and Lactobacillus spp. in breast-fed and Antibiotics, birth mode, and diet shape microbiome maturation
development of the bacterial flora in normal neonates. J. Med. formula fed infants as assessed by 16S rDNA sequence during early life. Sci. Transl Med. 8, 343ra82 (2016) | Chu, D. M. et al.
Microbiol. 14, 51–62 (1981). | Tompkins, A.M. et al. Diet and the differences. Microb. Ecol. Health Dis. 14, 97–105 (2002) | Palmer, C., Maturation of the infant microbiome community structure and
faecal microflora of infants, children and adults in rural Nigeria Bik, E. M., DiGiulio, D. B., Relman, D. A. & Brown, P. O. Development function across multiple body sites and in relation to mode of
and urban U.K. J. Hyg. 86, 285–293 (1981). | Daoulas Le of the human infant intestinal microbiota. PLoS Biol. 5, e177 (2007) | delivery. Nat. Med. 23, 314–326 (2017) | Wampach, L. et al.
Bourdelles, F., Avril, J. L. & Ghnassia, J. C. Quantitative study of the Bennet, R. & Nord, C. E. Development of the fecal anaerobic Colonization and succession within the human gut microbiome by
faecal flora of breast- or bottle-fed neonates (transl.). Arch. Fr. microflora after cesarean section and treatment with antibiotics archaea, bacteria and microeukaryotes during the first year of life.
Pediatr. 38, 35–39 (1981). in newborn infants. Infection 15, 332–336 (1987) | Dominguez- Front. Microbiol. 8, 738 (2017) | Stewart, C. J. et al. Temporal
FURTHER READING Tamburini, S., Shen, N., Wu, H. C. & Bello, M.G. et al. Delivery mode shapes the acquisition and development of the gut microbiome in early childhood from the
Clemente, J. C. The microbiome in early life: implications for structure of the initial microbiota across multiple body habitats in TEDDY study. Nature 562, 583–588 (2018) | Vatanen, T. et al.
health outcomes. Nat. Med. 22, 713–722 (2016) | Robertson, R. C., newborns. Proc. Natl Acad. Sci. USA 107, 11971–11975 (2010) | Genomic variation and strain-specific functional adaptation in the
Manges, A. R., Finlay, B. B. & Prendergast, A. J. The Human Bäckhed, F. et al. Dynamics and stabilization of the human gut human gut microbiome during early life. Nat. Microbiol. 4, 470–479
microbiome and child growth: first 1,000 days and beyond. Trends microbiome during the first year of life. Cell Host Microbe 17, (2018) | Yassour, M. et al. Strain-level analysis of mother-to-child
Microbiol. 27, 131–147 (2019) | Cooperstock, M. S. & Zedd, A. J. in 690–703 (2015) | Dominguez-Bello, M. G. et al. Partial restoration bacterial transmission during the first few months of life. Cell Host
Human Intestinal Microflora in Health and Disease (ed. Hentges, of the microbiota of cesarean-born infants via vaginal microbial Microbe 24, 146–154 (2018) | Ferretti, P. et al. Mother-to-infant
D. J.) Ch. 4 (Elsevier, 1983) | Tissier, H. Recherches sur la flore transfer. Nat. Med. 22, 250–253 (2016) | Jakobsson, H.E. et al. microbial transmission from different body sites shapes the
intestinale des nourrissons (état normal et pathologique) (Carre, G. Decreased gut microbiota diversity, delayed Bacteroidetes developing infant gut microbiome. Cell Host Microbe 24, 133–145
& C. Naud, C., Paris, 1900) | Long, S. S. & Swenson, R. M. colonization and reduced TH1 responses in infants delivered by (2018) | Wampach, L. et al. Birth mode is associated with earliest
Development of anaerobic faecal flora in healthy newborn cesarean section. Gut 63, 559–566 (2014) | Koenig, J. E. et al. strain-conferred gut microbiome functions and
infants. J. Pediatr. 91, 298–301 (1977) | Simhon, A., Douglas, J. R., Succession of microbial consortia in the developing infant gut immunostimulatory potential. Nat. Commun. 9, 5091 (2018) |
Drasar, B.S. & Soothill, J. F. Effect of feeding on infants’ faecal flora. microbiome. Proc. Natl Acad. Sci. USA 108, 4578–4585 (2011) | Lim, Aagaard, K. et al. The placenta harbors a unique microbiome. Sci.
Arch. Dis. Child 57, 54–58 (1982) | Stark, P. L. & Lee, A. The microbial E. S. et al. Early-life dynamics of the human gut virome and Transl. Med. 6, 237ra265 (2014) | Kliman, H.J. Comment on “The
ecology of the large bowel of breast-fed and formula-fed infants bacterial microbiome in infants. Nat. Med. 21, 1228–1234 (2015) | placenta harbors a unique microbiome”. Sci. Transl. Med. 6, 254le4
during the first year of life. J. Med. Microbiol. 15, 189–203 (1982) | Yatsunenko, T. et al. Human gut microbiome viewed across age (2014) | Subramanian, S. Persistent gut microbiota immaturity in
Satokari, R., Vaughan, E. E., Favier, C., Edwards, C. & de Vos, W. M. and geography. Nature 486, 222–227 (2012) | Bokulich, N. A. et al. malnourished Bangladeshi children. Nature 510, 417–421 (2014).

MILESTONES
M I L E S TO N E 6 accurate and complete assembly of genome

sequences is still hampered by the difficulty
Sequence-based identification in resolving long repetitive regions. Thus, ref-

erence-based sequencing methods emerged.
Concerted sequencing efforts have been
of human-associated microbiota made to construct microbial reference
sequences. In 2007, the National Institutes
of Health launched the Human Microbiome
Project and, five years later, published the
first reference data for microorganisms
collected from 242 healthy United States
Credit: Sean Prior / Alamy Stock Photo
volunteers, covering a number of anatomical

sites such as mouth, nose, skin, lower intes-
tine and vagina (MILESTONE 17). The valuable
genome references that were generated by
this consortium allow reliable identification
of individual microbial species, but fall
short when new genomes are present in the
community.
Long-read sequencing offers an alternative
solution for mapping challenging repetitive
Over the past twenty years, advances in cloning step and is prohibitively expensive for regions. For example, single molecule, real-
DNA sequencing technology have deepened large-scale microbiome studies. The advent time (SMRT) DNA sequencing, in combina-
our knowledge in every niche of biology, of next generation sequencing (NGS) offered tion with a short-read shotgun DNA library,
from annotation of the human genome to a cost-effective method that eliminated allows for de novo microbial genome assem-
sequencing the human-associated microbial the cloning step by amplifying 16S rRNA blies, although it suffers from high error rates.
metagenome, the genetic material of the genes using primers containing sequencing Access to genome sequencing has
microorganisms that inhabit almost every adapters and barcodes. The massive parallel transformed human microbiome research
surface of the human body. sequencing throughput offered by NGS has from focusing on identity characterizations,
Study of the human microbiota histori- significantly increased the sequence depth of to metagenomics approaches that not only
cally relied on culture-dependent methods to 16S rRNA genes, allowing for taxonomic and reveal microbial species but also how micro-
isolate and grow bacterial colonies in a prede- phylogenetic analyses of complex microbial bial metabolic activities correlate with human
termined medium. The inability to cultivate communities. Yet, 16S rRNA sequencing can- health and disease.
a large portion of microorganisms, however, not always resolve closely related species and In addition to metagenomics, meta-
substantially underestimated the biodiversity may miss the intra-species diversity. To better transcriptomic analysis enabled by RNA
of human-associated microbial communities. capture the full picture, shotgun sequencing sequencing offers a way to detect active
An early milestone in this field was was developed for direct sequencing of DNA. members present in a microbial community
the adoption of a technology, previously The advantage is its capability to recover the (MILESTONE 14).
pioneered by Carl Woese, Norman Pace and underrepresented microorganisms that were The construction of metagenome-assem-
others to identify environmental bacteria, often masked by high-abundance species. bled genomes, an approach also pioneered
that was based on sequencing small subunit Shotgun sequencing can use short-read in environmental microbiology and recently
ribosomal RNA genes (16S rRNA). Using this (such as Illumina) or long-read (for example, applied to human-associated communi-
approach, Wilson and Blitchington compared Oxford Nanopore MinION and Pacific ties, will provide a new, unprecedented
the diversity of cultivated and noncultivated Bioscience Sequel) platforms. The short-read opportunity for deep characterization of the
bacteria within a human faecal sample in approach typically demands extensive com- functional potential of the human microbial
1996. Since then, sequencing of 16S rRNA putational support for assembling short reads ecosystem.
genes from complex communities has become into meaningful sequences. However, an Lei Tang, Nature Methods
a powerful tool for assessing microbial
diversity in the human microbiota. In 2005, ORIGINAL ARTICLES Wilson, K. H. & Blitchington, R. B. Human by 16S rDNA sequence analysis. FEMS Microbiol. Ecol. 39, 33–39
colonic biota studied by ribosomal DNA sequence analysis. Appl. (2002) | Pei, Z. et al. Bacterial biota in the human distal
Eckburg et al. analysed samples not only from Environ. Microbiol. 62, 2273–2278 (1996) | Suau, A. et al. Direct esophagus. Proc. Natl Acad. Sci. USA 101, 4250–4255 (2004) |
faeces, but also from multiple colonic mucosal analysis of genes encoding 16S rRNA from complex Bik, E. M. et al. Molecular analysis of the bacterial microbiota in
sites. They sequenced >13,000 16S rRNA communities reveals many novel molecular species within the the human stomach. Proc. Natl Adad. Sci. USA 103, 732–737
human gut. Appl. Environ. Microbiol. 65, 4799–4807 (1999) | (2006) | Fredricks, D. N. Molecular identification of bacteria
genes, which constituted a substantial increase Kroes, I., Lepp, P. W. & Relman, D. A. Bacterial diversity within associated with bacterial vaginosis. N. Engl. J. Med. 353,
in scope over previous work, and discovered the human subgingival crevice. Proc. Natl Acad. Sci. USA 96, 1899–1911 (2005) | Hyman, R. W. et al. Microbes on the human
14547–14552 (1999) | Eckburg, P. B. et al. Diversity of the human vaginal epithelium. Proc. Natl Acad. Sci. USA 102, 7952–7957
significant inter-subject variability and
intestinal microbial flora. Science 308, 1635–1638 (2005) | (2005) | Gao, Z., Pei, Z., Tseng, C.-H. & Blaser, M. J. Molecular
greater differences between stool and mucosal Gill, S. R. et al. Metagenomic analysis of the human distal gut analysis of human forearm superficial skin bacterial biota. Proc.
community composition than previously microbiome. Science 312, 1355–1359 (2006). Natl Acad. Sci. USA 104, 2927–2932 (2007) | Grice, E. A. et al.
FURTHER READING Relman, D. A., Schmidt, T. M., Topographical and temporal diversity of the human skin
described. MacDermott, R. P. & Falkow, S. Identification of the uncultured microbiome. Science 324, 1190–1192 (2009) | Human
Sequencing of marker genes (such as 16S bacillus of Whipple’s disease. N. Engl. J. Med. 327, 293–301 Microbiome Jumpstart Reference Strains Consortium et al.
rRNA) is traditionally associated with Sanger (1992) | Hold, G. L., Pryde, S. E., Russell, V. J., Furrie, E. & Flint, H. A catalog of reference genomes from the human microbiome.
J. Assessment of microbial diversity in human colonic samples Science 328, 994–999 (2010).
sequencing, which requires a labour-intensive

MILESTONES
M I L E S TO N E 7 from 576.6 gigabases of metagenomic sequences

from the faecal samples of 124 individuals.
Stability and individuality of

These genes were found to be largely shared by
individuals of the cohort, and 18 species were
detected in all individuals. However, a key study
adult microbiota that examined the faecal microbiomes of six

adult twin pairs and their mothers suggested
that there was an identifiable core microbiome at
A plethora of studies conducted over the past the gene level rather than the microbial species
few decades have revealed strong associations level. In this study, individuals shared >93% of the
between a disrupted microbiota and diseases, for enzyme-level functional groups, but no bacterial
example, inflammatory bowel disease. However, phylotypes were present at >0.5% in all samples.
the key to understanding the role of a disrupted Many of these concepts were subsequently
microbiota in human diseases is first to answer confirmed in large-population studies published
the question: what is a ‘normal’ microbiota? by the Human Microbiome Project (HMP)
This question still frustrates many Consortium. Analysis of samples collected
microbiologists, even with the advent of from 242 healthy adults from up to 18
high-throughput sequencing and ’omics body sites showed that each habitat is
techniques. Prior to the availability of characterized by a small number of highly
these technologies, however, several abundant signature taxa, but that the relative
studies were instrumental in answering similar abundance of taxa and genes in each habitat
questions to help define ‘normal’, such as how varies between individuals.
much microbial variation is there between and One drawback to the HMP dataset is the
within adults, and is the microbiota stable? limited temporal scope. Instead, other studies
In 1998, when conventional microbiological have shed further light on the stability of the
techniques involving plate count analyses adult microbiota. For example, in one study,
had reached an impasse in what they could a human microbiota time series was obtained
reveal about human microbial diversity, covering two individuals at four body sites over
molecular approaches were instead beginning 396 time-points (daily for up to 15 months).
to be implemented. A study by Willem de Vos Credit: Zdeněk Malý / Alamy Stock Photo Despite finding stable differences between
and colleagues used polymerase chain reaction body sites and individuals, this high-resolution
amplification of regions of the 16S ribosomal diversity. Other studies dedicated to skin and temporal analysis did show pronounced
(r)RNA gene, which is often used to infer the vaginal microbiomes were also key to our variability in an individual’s microbiota across
genetic relationships between organisms, and understanding of an individualised microbiota. It months, weeks and days. In another study, an
then temperature gradient gel electrophoresis was found that comparable skin sites had similar analysis of the faecal microbiota of 37 individuals
(TGGE) to visualize the diversity of the amplified bacterial communities, but that the complexity found that ~60% of bacterial strains remained
gene. Comparisons of the banding profiles and temporal stability of the communities were stable for up to five years.
generated by TGGE from 16 adult faecal samples site-dependent, whereas other studies found Overall, samples obtained from the same
indicated that each individual has their own that the vaginal microbiome differs among individual are more similar to one another than
unique microbial community. Furthermore, individuals and, markedly, change over a short those from different individuals, suggesting
by monitoring two individuals over time, the time. Together, these studies revealed that the each person has a microbiota that is distinct
researchers showed that the TGGE profiles were human microbiota is highly variable both within and stable. Much is still unknown regarding how
stable over a period of at least six months. and between individuals. stable the microbiota is to perturbations, such
Similar molecular approaches were applied to One of the goals of many studies characterising as those arising from antibiotics, diet and the
different sites of the human body, with the goal the diversity and stability of the human immune system. However, further studies in-line
of improving our understanding of adult human microbiota was to establish whether there was with those discussed here will no doubt enhance
microbial diversity. In 2005, one study moved a core microbiota — are there bacterial species our view of human microbiota dynamics to
beyond using faecal microbiota as a surrogate that we all share? In 2010, the international ultimately understand what is ‘normal’.
for the entire gut microbiota and sampled MetaHIT (Metagenomes of the Human Intestinal Iain Dickson,
multiple colonic mucosal sites from three healthy Tract) project published a gene catalogue derived Nature Reviews Gastroenterology & Hepatology
individuals. Through an analysis of 13,335 16S
rRNA gene sequences, this work confirmed ORIGINAL ARTICLE Zoetendal, E. G. et al. Temperature long-term stability of the human gut microbiota. Science 341,
marked microbial variation between individuals gradient gel electrophoresis analysis of 16S rRNA from human 1237439 (2013) | Rajilić-Stojanović, M. et al. Long-term monitoring
faecal samples reveals stable and host-specific communities of of the human intestinal microbiota composition. Env. Microbiol. 15,
and showed that the adult gut mucosal
active bacteria. Appl. Environ. Microbiol. 64, 3854–3859 (1998) 1146–1159 (2013) | Schloissnig, S. et al. Genomic variation
microbiota was dominated by Bacteroidetes FURTHER READING Eckburg, P. B. et al. Diversity of the human landscape of the human gut microbiome. Nature 493, 45–50 (2013) |
and Firmicutes, whereas Actinobacteria, intestinal microbial flora. Science 308, 1635–1638 (2005) | Costello, Lahti, L., Salojarvi, J., Salonen, A., Scheffer, M. & de Vos, W. M.
E. K. et al. Bacterial community variation in human body habitats Tipping elements in the human intestinal ecosystem. Nat. Comm. 5,
Proteobacteria and Verrucomicrobia were
across space and time. Science 326, 1694–1697 (2009) | Grice, E. A. 4344 (2014) | DiGiulio, D. B. et al. Temporal and spatial variation of
relatively minor constituents. et al. Topographical and temporal diversity of the human skin the human microbiota during pregnancy. Proc. Natl Acad. Sci. USA
Another study went one step further, in microbiome. Science 324, 1190–1192 (2009) | Arumugam, M. et al. 112, 11060-5 (2015) | Lloyd-Price, J. et al. Strains, functions and
2009, and examined bacterial diversity of 27 Enterotypes of the human gut microbiome. Nature 473, 174–180 dynamics in the expanded Human Microbiome Project. Nature 550,
(2011) | Wu, G. D. et al. Linking long-term dietary patterns with gut 61–66 (2017) | Mehta, R. S. et al. Stability of the human faecal
body sites from at least seven individuals and at microbial enterotypes. Science 334, 105–108 (2011) | Caporaso, J. G. microbiome in a cohort of adult men. Nat. Microbiol. 3, 347–355
four different time points. High interpersonal et al. Moving pictures of the human microbiome. Genome Biol. 12, (2018) | Sommer, F., Anderson, J. M., Bharti, R., Raes, J. & Rosenstiel, P.
variability was found across all body sites but R50 (2011) | Gajer, P. et al. Temporal dynamics of the human vaginal The resilience of the intestinal microbiota influences health and
microbiota. Sci. Transl Med. 4, 132ra52 (2012) | Faith, J. J. et al. The disease. Nat. Rev. Microbiol. 15, 630–638 (2017).
individuals experienced minimal temporal

MILESTONES
fungal community of humans) has also been

Credit: P. Patenall / Springer Nature Limited
fruitfully studied. A number of recent studies

have highlighted the crucial roles of fungi in
both healthy and disease states. For example,
enteric commensal bacteria and fungi may have
redundant protective and tolerizing functions in
the context of regulating the immune response,
suggesting that gut homeostasis can be retained
through the mycobiome even in the absence of
‘good’ bacteria. In inflammatory bowel diseases,
however, mycobiome dysbiosis contributes to
M I L E S TO N E 8 disease progression, with fungi enriched at the
expense of bacteria. These data, along with other
Beyond bacteria: studies of other studies, suggest a complex interplay between

fungi and bacteria.
host-associated microorganisms Recently, the human archaeome (the

community of human-associated archaea)
has attracted interest due to a number of
While bacteria are a major component of the Viral metagenomics has continued to advance, observations, including recognition of the human
human microbiota, viruses, fungi and archaea with higher-throughput techniques enabling gut archaeon Methanosphaera stadtmanae by the
are also important members of the community, more rapid virus discovery and classification in immune system and the discovery of previously
with potential effects on human health. The both healthy and diseased tissues, and helping undetected human-associated archaea.
development of 16S ribosomal RNA gene us to understand the role of commensal and Methanogenic archaea are amongst the most
sequencing transformed the field of microbiome pathogenic viruses in the context of the wider abundant microorganisms in the human gut and
research by enabling bacterial and archaea microbiome. sometimes outnumber even the most abundant
phylogenetic analyses without the need for In a study published in 2010, by Jeffrey Gordon bacterial species. In 2004, methanogenic archaea
culturing (MILESTONE 6). Fungi can also be and Rowher, faecal viromes of healthy adult were found to be associated with the onset
identified and classified by sequencing a common female twins and their mothers were shown to be of periodontal disease, identifying the first
nuclear ribosomal internal transcribed spacer individually distinct and stable over the course link between archaea and human disease. As
region. Viruses, however, are far more challenging of a year, in agreement with faecal bacterial data with bacteria, viruses and fungi, archaea have
to isolate and sequence due to the necessity from this same cohort. Other studies have begun been isolated from various human anatomical
of a eukaryotic or prokaryotic host and the to elucidate the effects of disease and diet on sites, including the gut, skin, vagina and oral
absence of conserved genes. The modern era of the gut virome, finding that phage composition cavity. Yet, owing to fundamental differences in
human-associated viral metagenomics actually became increasingly similar between individuals biology, they have often remained undetected
started in the ocean. In 2001, the research group on the same diet, and that significant expansion in microbiome surveys, warranting further
of Forest Rowher, a marine microbial ecologist of one phage order is associated with Crohn’s investigation of the human archaeome and the
at San Diego State University, published disease and ulcerative colitis. Viral microbiome role of archaea in human health and disease.
a randomized shotgun library sequencing signatures may therefore be associated with As identification and characterization of the
method to analyse genomic DNA from a single environmental influences as well as disease human virome, mycobiome and archaeome in
bacteriophage. This was rapid, reasonably progression, including in tissues beyond the gut. various tissues and disease states continues
unbiased and required very little DNA input, Shotgun metagenomics has facilitated a more to improve, it will be increasingly important
a crucial limiting variable for sequencing. The functional and interconnected perspective to delineate function and how these other
research team further demonstrated the utility of of the microbiome, with the contributions of ‘omes’ interact as a community to preserve or
this technique beyond single phage analysis by fungi and archaea also becoming increasingly dysregulate human health.
characterizing the more complex viral make-up understood. The human mycobiome (the Saheli Sadanand, Nature Medicine
of seawater and marine sediment, paving the
way for analyses of human viromes and a deeper
ORIGINAL ARTICLE Breitbart, M. et al. Metagenomic analyses disease. mBio 7, e01250-16 (2016) | Sokol, H. et al. Fungal
characterisation of the human microbiome. of an uncultured viral community from human feces. J. Bacteriol. microbiota dysbiosis in IBD. Gut 66, 1039–1048 (2017) | Lepp, P. W.
By 2003, bacteriophages that infected individual 185, 6220–6223 (2003). et al. Methanogenic Archaea and human periodontal disease.
bacterial species had been identified from FURTHER READING Schoch, C. L. et al. Nuclear ribosomal Proc. Natl Acad. Sci. USA 101, 6176–6181 (2004) | Koskinen, K. et al.
internal transcribed spacer (ITS) region as a universal DNA First insights into the diverse human archaeome: specific
human faecal waste, but the diversity and relative barcode marker for Fungi. Proc. Natl Acad. Sci. USA 109, 6241– detection of archaea in the gastrointestinal tract, lung, and nose
abundance of different phages remained unknown, 6246 (2012) | Rowher, F. et al. Production of shotgun libraries using and on skin. mBio 8, e00824-17 (2017) | Vierbuchen, T. et al. The
as existing approaches were biased towards random amplification. Biotechniques 31, 108–118 (2001) | Reyes human-associated archaeon Methanosphaera stadtmanae is
et al. Viruses in the faecal microbiota of monozygotic twins and recognized through its RNA and induces TLR8-dependent NLRP3
bacteria already known to be infected by phages.
their mothers. Nature 466, 334–338 (2010) | Norman, J. M. et al. inflammasome activation. Front. Immunol. 8, 1535 (2017) |
By using the linker-amplified shotgun library Disease-specific alterations in the enteric virome in inflammatory Borrel, G. et al. Genomics and metagenomics of trimethylamine-
approach, Rowher’s research group provided the bowel disease. Cell 160, 447–460 (2015) | Minot, S. et al. The utilizing Archaea in the human gut microbiome. ISME J. 11, 2059–
human gut virome: inter-individual variation and dynamic 2074 (2017) | Adam, P. S. et al. The growing tree of Archaea: new
first quantitative description of the composition of
response to diet. Genome Res. 21, 1616–1625 (2011) | Jiang, T. T. perspectives on their diversity, evolution and ecology. ISME J. 11,
the uncultured virome in human faeces collected et al. Commensal fungi recapitulate the protective benefits of 2407 (2017) | Nottingham, P. M. et al. Isolation of methanogenic
from a single healthy adult. The majority of phage intestinal bacteria. Cell Host Microbe 22, 809–816 (2017) | Shao, T. Y. bacteria from feces of man. J. Bacteriol. 96, 2178–2179 (1968) |
sequence matches were from temperate phages, et al. Commensal Candida albicans positively calibrates systemic Breitbart, M. et al. Viral diversity and dynamics in an infant gut.
Th17 immunological responses. Cell Host Microbe 25, 404–417 Res. Microbiol. 159, 367–373 (2008) | Ghannoum, M. A. et al.
which commonly integrate into the host bacterial (2019) | Iliev, I. D. et al. Interactions between commensal fungi and Characterization of the oral fungal microbiome (mycobiome) in
genome. The faecal virome was also dominated by the C-type lectin receptor Dectin-1 influence colitis. Science 336, healthy individuals. PLoS Pathog. 6, e1000713 (2010) | Yang, A. M.
phages known to infect Gram-positive bacteria, in 1314–1317 (2012) | Hoarau, G. et al. Bacteriome and mycobiome et al. Intestinal fungi contribute to development of alcoholic liver
interactions underscore microbial dysbiosis in familial Crohn’s disease. J. Clin. Invest. 127, 2829–2841 (2017).
keeping with prior data on faecal bacterial content.

MILESTONES

M I L E S TO N E 9
Regulation of mucosal
immunity by the microbiota
The relationship between the immune system In 2004, Seth Rakoff-Nahoum and Ruslan
and microorganisms was long viewed as a Medzhitov provided evidence that the immune
war rather than a union, as it was mostly system senses commensals through PRRs
studied in the context of host defence against under normal conditions and that this sensing
pathogens. Thus, when deficiency in lym- is crucial for tissue repair. This finding opened
phoid organ development and immune cell a new perspective on immune response to
activity was reported in germ-free animals in microorganisms not as host defence, but as a
the 1960s, this first evidence that microbiota symbiotic physiological process.
shape immune homeostasis was interpreted Realization of beneficial roles of immune–
as education of the immune system by microbial interactions prompted a revision
infections. of the hygiene hypothesis to postulate that
This concept laid the foundation for protection from allergic diseases is mediated
the ‘hygiene hypothesis’, formalized by by early-life exposure to commensals rather
David Strachan in 1989 in his influential than pathogens (MILESTONE 5). The refined
study reporting lower incidence of hay hypothesis suggested that the rise of allergies
fever and eczema in children with older in industrialized societies is caused by loss
siblings. He proposed that infections in early of commensals. A link between lifestyle,
childhood prevent atopy later in life, and that microbiota and allergic diseases has been
increased allergy prevalence in developed now confirmed in a large number of human
countries may be caused by high standards of observational studies, and expanded to Kenya Honda, Dan Littman and colleagues
personal hygiene. Further developments of implicate microbiota in other chronic inflam- uncovered how specific commensal bac-
the hygiene hypothesis correlated pathogen matory and autoimmune pathologies, as well teria direct Th17 cell differentiation in the
exposure to decreased allergy risk in humans, as metabolic and neurological disease. small intestine. In 2013–2014, four groups
and established a role for microbiota in oral Dissecting the underlying cellular and independently discovered a mechanism of
tolerance in mice, presumably in shifting an molecular complexity of microbiota–immune immune tolerance mediated by metabolites
immune set point from T helper 2 (Th2) to interactions has occupied microbiologists and of intestinal commensals (MILESTONE 21).
Th1 cell responses. immunologists to this day. Pioneering works Our intimate companion in sickness and
In parallel, fundamental principles of by Sarkis Mazmanian, Dennis Kasper and in health, microbiota impacts our physiol-
microorganism recognition by the immune colleagues showed that microbiota-guided ogy to a large extent through interactions
system unfolded. In 1989, Charles Janeway maturation of the mouse immune system can with the immune system at mucosal sites.
proposed that immune responses are initiated be recapitulated by a polysaccharide produced How these interactions, ranging from host
by genome-encoded pattern recognition by a symbiotic bacterium, and delineated defence to active tolerance to symbiosis,
receptors (PRR) on immune cells, which sense that its uptake by dendritic cells promotes integrate into physiological outcomes
conserved microbial molecules. Over the next antigen presentation, inducing expansion remains an exciting avenue to explore.
decade, PRRs specific to various bacterial, and differentiation of CD4+ T cells into Th1 Tanya Bondar,
fungal and viral components were discovered. and regulatory T cell lineages. Ivaylo Ivanov, Nature Communications
However, it became evident that PRRs are not
specific to pathogens, posing the question of ORIGINAL ARTICLES Strachan, D. P. Hay fever, hygiene, and Symbiotic bacteria direct expression of an intestinal bactericidal
household size. BMJ 299, 1259–1260 (1989) | Rakoff-Nahum, S. lectin. Science 313, 1126–1130 (2006) | Mazmanian, S. K., Round,
how commensals, which colonize mucosal et al. Recognition of commensal microflora by toll-like receptors J. L., & Kasper, D. L. A microbial symbiosis factor prevents
surfaces, coexist with the immune system. is required for intestinal homeostasis. Cell 118, 229–241 (2004) | intestinal inflammatory disease. Nature 453, 620–625 (2008) |
The prevailing explanation was that com- Mazmanian, S. K. et al. An immunomodulatory molecule of Ivanov, I. I. et al. Specific microbiota direct the differentiation of
symbiotic bacteria directs maturation of the host immune IL-17-producing T-helper cells in the mucosa of the small
mensals and immune cells are separated by system. Cell 122, 107–118 (2005). intestine. Cell Host Microbe 4, 337–349 (2008) | Wen, L. et al.
epithelial barriers. This view was challenged in FURTHER READING Sudo, N. et al. The requirement of Innate immunity and intestinal microbiota in the development of
the early 2000s by accumulating examples of intestinal bacterial flora for the development of an IgE Type 1 diabetes. Nature 455, 1109–1113 (2008) | Hall, J. A. et al.
production system fully susceptible to oral tolerance induction. Commensal DNA limits regulatory T cell conversion and is a
immune responses to commensals in healthy J. Immunol. 159, 1739–1745 (1997) | Macpherson A. J. et al. A natural adjuvant of intestinal immune responses. Immunity 29,
mice. Among these, Andrew Macpherson, primitive T cell-independent mechanism of intestinal mucosal 637–649 (2008) | O’Mahony, C. et al. Commensal-induced
IgA responses to commensal bacteria. Science 288, 2222–2226 regulatory T cells mediate protection against pathogen-
Rolf Zinkernagel and colleagues found that (2000) | Hooper, L. et al. Angiogenins: a new class of microbicidal stimulated NF-kappaB activation. PLoS Pathog. 4, e1000112
in mice, commensals are recognized and proteins involved in innate immunity. Nat. Immunol. 4, 269–273 (2008) | Ivanov, I. I. et al. Induction of intestinal Th17 cells by
compartmentalized by gut lumen-secreted (2003) | Bashir, M. E. et al. Toll-like receptor 4 signaling by segmented filamentous bacteria. Cell 139, 485–498 (2009) |
intestinal microbes influences susceptibility to food allergy. Wu, H.-J. et al. Gut-residing segmented filamentous bacteria
IgA. Lora Hooper, Jeffrey Gordon and cow- J. Immunol. 172, 6978–6987 (2004) | Mazmanian, S. K. et al. An drive autoimmune arthritis via T helper 17 cells. Immunity 32,
orkers reported that a commensal bacterium immunomodulatory molecule of symbiotic bacteria directs 815–827 (2010) | Naik, S. et al. Compartmentalized control of skin
stimulates antimicrobial peptide production maturation of the host immune system. Cell 122, 107–118 (2005) | immunity by resident commensals. Science 337, 1115–1119
Cash, H. L., Whitham, C. V., Behrendt, C. L. & Hooper, L. V. (2012).
by Paneth cells.

MILESTONES
M I L E S TO N E 1 0
The importance of
feeding your microbiota
Most complex plant polysaccharides enzymes dependent on food source,
Springer Nature Limited

are not digested by humans and enter exposing a flexible network of
Credit: P. Patenall /
the colon as a potential food source genes for harvesting glycans
for the microbiota. But, in the late based on their availability in
1970s, the extent to which gut bacte- the host.
ria could metabolize this dietary fibre Subsequent work on
was largely unknown. B. thetaiotaomicron elucidated
To bridge this gap, Abigail Salyers many of the gene clusters and
and colleagues tested the ability of a pathways involved in polysaccharide
wide range of anaerobic bacterial spe- metabolism that enable bacteria to
cies resident in the human colon to use the diversity of glycans provided
ferment plant polysaccharides as well by a mammalian diet and the host
as intestinal mucins (glycosylated itself. This ability to harvest host gly- mice suggest they may. A fibre-free
proteins that line the gut epithelium). cans, such as during fluctuations of diet in mice reduced the thickness of
They found that the bacterial strains a host’s diet, was shown to critically Such findings the colonic mucus layer, as predicted,
had a diverse and inducible ability to affect survival of B. thetaiotamicron and increased susceptibility to dis-
[…] underscore
break down different substrates, with in mono-colonized mice fed a fibre- ease caused by a mouse enteric path-
the largest variety of polysaccharides free diet. Such findings, as well as the important ogen. In another study, the microbial
fermented by Bifidobacterium and later studies, underscore the impor- interplay production of short chain fatty acids
Bacteroides species. The researchers tant interplay of host diet and glycan of host diet from dietary fibre influenced mouse
proposed that by altering availability metabolism for gut colonization of lung disease and immune responses.
of preferred bacterial food sources in human commensals and their persis-
and glycan And microbial metabolism of other
the host diet — such as limiting fibre tence in populations over time. metabolism components of the human diet, such
intake — could trigger induction We now know that gut micro- for gut as L-carnitine in meat, has been
of enzymes capable of degrading organisms harbour thousands of colonization linked to atherosclerosis.
the intestinal mucin layer, affecting genes involved in catabolism of Much remains to be understood
human health and even colon cancer. carbohydrates, but how this enzy- of human of the impact of diet and the micro-
But it was not until 2005 that matic breadth was acquired remains commensals biota on human health and disease.
Jeffrey Gordon’s group demonstrated unclear. One potential source of But these studies shed light on the
that a change in diet in a mammal genetic diversity is horizontal transfer dynamic effect of altering host diet
could alter the degradative activity of genes from environmental micro- on the makeup and behaviour of
of the colonic microbiota in vivo. By organisms to gut bacteria. In a search microorganisms in the gut and other
colonizing the gut of germ-free mice for enzymes expressed by a marine organs, with potential implications for
with Bacteroides thetaiotaomicron Bacteroidetes that degrade sulfated disease modification and treatment.
— which Salyers and colleagues polysaccharides found in edible Alison Farrell, Nature Medicine
had identified as a human symbiont seaweed (such as nori), porphyranases
capable of fermenting a wide range of were identified that had a homologue ORIGINAL ARTICLE Sonnenburg, J. L. et al. Glycan foraging in vivo by an intestine-
glycan substrates — the researchers in a gut bacterium, Bacteroides ple- adapted bacterial symbiont. Science 307, 1955–1959 (2005).
FURTHER READING Salyers, A. A. et al. Fermentation of mucin and plant
tested, in a physiologically relevant beius. Additional porphyranases and
polysaccharides by strains of Bacteroides from the human colon. Appl. Environ. Microbiol.
setting, the effects of different diets agarases were subsequently identified 33, 319–322 (1977) | Salyers, A. A. Fermentation of mucins and plant polysaccharides by
on expression of bacterial genes. in the microbiomes of Japanese but anaerobic bacteria from the human colon. Appl. Environ. Microbiol. 34, 529–533 (1977) |
Salyers A. A. et al. Degradation of polysaccharides by intestinal bacterial enzymes. Am.
In mice fed a fibre-rich diet, not North American individuals. The J. Clin. Nutr. 31, 128–130 (1978) | Martens, E. C. et al. Mucosal foraging enhances fitness
B. thetaiotaomicron genes involved findings suggest that transfer of genes and transmission of a saccharolytic human gut bacterial symbiont. Cell Host Microbe 4,
in polysaccharide metabolism were from marine bacteria on nori was the 447–457 (2008) | Sonnenburg, E. D. et al. Diet-induced extinctions in the gut microbiota
compound over generations. Nature 529, 212–215 (2016) | Hehemann, J. H. et al. Transfer
significantly upregulated compared likely origin of enzymes in the human of carbohydrate-active enzymes from marine bacteria to Japanese gut microbiota.
to their expression in bacteria grown gut that diversify the ability of bacteria Nature 464, 908–912 (2010) | Desai, M. S. et al. A dietary fiber-deprived gut microbiota
degrades the colonic mucus barrier an enhances pathogen susceptibility. Cell 167,
in a minimal medium. By contrast, to harvest energy from food sources,
1339–1353 (2016) | Trompette, A. et al. Gut microbiota metabolism of dietary fiber
in mice fed a diet devoid of complex such as algal polysaccharides in influences allergic airway disease and hematopoiesis, Nature Med. 20, 159–166 (2014) |
polysaccharides, the most highly seaweed, a major dietary component Koeth, R. A. et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat,
promotes atherosclerosis. Nature Med. 19, 576–585 (2013) | Maldonado- Gómez, M. X. et
upregulated bacterial genes were those in Japan. al. Stable engraftment of Bifidobacterium longum AH1206 in the human gut depends on
involved in host glycan degradation. But do the nutrient foraging individualized features of the resident microbiome. Cell Host Microbe 20, 515–526 (2016)
The work confirmed in a vertebrate properties of gut microbiota have | Shepherd, E. S. et al. An exclusive metabolic niche enables strain engraftment in the
gut microbiota. Nature 557, 434–438 (2018).
the differential expression of bacterial a role in human health? Results in

MILESTONES
More recently, the intestinal micro-

M I L E S TO N E 1 1 biota has been shown to modulate
neurological and psychiatric diseases,
Transfer of host phenotypes through as well as cancer, adding to those
phenotypes that can be transferred
microbiota transplantation by faecal material or mother-to-child
transmission. Intestinal tumours can
Chronic inflammatory conditions, be induced in mice harbouring an
such as obesity, diabetes, heart dis- oncogenic mutation in Kras with a
ease, autoimmune disorders and can- HFD, independent of obesity, and this
cer, have long been associated with effect can be transferred to healthy
a Westernized diet. Environmental adult, normal-diet, Kras mutant mice
factors also play a role, with the gut via the dysbiotic gut microbial com-
microbiota taking centre stage in the munity found in the faecal material
past two decades. Indeed, high-fat of HFD mice. Inflammation-induced
diets (HFDs) regulate microbial colorectal cancer is transferrable
communities in drastic fashion. The to co-housed mice, an effect that is
microbiota is now acknowledged as blocked by antibiotic treatment of
having direct, even causative roles in donor mice. The impact of gut micro-
mediating connections between organisms on promoting obesity and
the environment, food intake and liver cancer induced by a Westernized
chronic disease. diet is trans-generational — it can
Early studies using germ- be seen in the progeny as well as
free mice showed that body fat the grandchildren of mother mice
content and insulin resistance consuming the diet, even though the
are transferable from obese to lean mothers themselves are unaffected
mice through exposure to faecal by these conditions. Finally, the
material. In a pioneering paper, intestinal microbiota influences brain
Credit: Gl0ck / Alamy Stock Photo
researchers found that the microbiota development and function in mice.
of obese mice are more efficient For instance, distinct anxiety levels of
at extracting energy from the host These findings have raised the different mouse strains are linked to
diet compared to the microbiota possibility of developing therapies distinct microbiota compositions and
of lean ones and that increased based on modulating the microbiota. These findings the phenotypes are transferrable via
adiposity is transferable. When One such proof-of-principle study have raised faecal matter.
the microbiota from the cecum of found that small intestinal infusion There is a large body of anecdotal
obese mice, which had a higher of the gut microbiota from a lean the possibility and direct evidence suggesting that
Firmicutes/Bacteroidetes ratio than donor restored insulin sensitivity of developing the microbiota has a role in the
lean donors, were transplanted into and increased the microbial diversity therapies health of many human functional
germ-free recipients, there was a (with concomitant short-chain fatty systems. Being able to transmit
based on
greater increase in body fat than in acid metabolism) in obese human phenotypes via whole microbiota,
recipients of microbiota from lean subjects with untreated metabolic modulating individual microorganisms or, even-
mice. Notably, the structure of the syndrome. This study also described the microbiota tually, through individual microor-
established colonizing community a role for butyrate from gut micro- ganism-derived metabolites, should
in the recipient was highly similar to bial metabolism in improving insulin prove revolutionary for treatment of
that of the original donor, suggesting sensitivity, a first step in defining disease and maintenance of a healthy
that the composition of the donor specific species and mechanisms human condition.
microbiota is critical for development used by gut microorganisms to Mirella Bucci, Nature Chemical Biology
of the obese phenotype. modulate host physiology. Extending
Subsequent studies using these findings beyond adiposity and ORIGINAL ARTICLE Turnbaugh, P.J. et al. An obesity-associated gut microbiome with
increased capacity for energy harvest. Nature 444, 1027–1031 (2006).
gnobiotic mice (with a defined micro- insulin resistance, host phenotypes FURTHER READING Bäckhed, F. et al. The gut microbiota as an environmental factor
biota) showed that a single endo were also found to be transferable that regulates fat storage. Proc. Natl Acad. Sci. USA 101 15718–15723 (2004) | Garrett,
toxin-producing Enterobacter species in mice by co-housing or through W.S. et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate
immune system. Cell 131, 33–45 (2007) | Vrieze, A. et al. Transfer of intestinal microbiota
isolated from an obese human’s gut vertical transmission. For instance, from lean donors increases insulin sensitivity in individuals with metabolic syndrome.
was sufficient to induce obesity, a colitis phenotype (resembling Gastroenterology 143, 913–916 (2012) | Fei, N. et al. An opportunistic pathogen from
the gut of an obese human causes obesity in germfree mice. ISME J. 7, 880–884 (2013) |
insulin-resistant phenotypes and human ulcerative colitis) can be
Schulz, M.D. et al. High-fat-diet-mediated dysbiosis promotes intestinal carcinogenesis
systemic inflammation in mice fed a induced by genetic mutation or can independently of obesity. Nature 514, 508–512 (2014) | Poutahidis, T. et al. Dietary
HFD. The results, derived from the be transferred, vertically to wildtype microbes modulate transgenerational cancer risk. Cancer Res. 75, 1197–1204 (2015) |
Collins, S. M. et al. The adoptive transfer of behavioural phenotype via the intestinal
first gnobiotic mouse obesity model, progeny or horizontally to wildtype microbiota: experimental evidence and clinical implications. Curr. Opin. Microbiol. 16,
also represent the first demonstration cage-mates, through co-housing 240–245 (2013) | Hu, B. et al. Microbiota-induced activation of epithelial IL-6 signaling
of a causative role of the microbiota in — a process that is blocked with links inflammasome-driven inflammation with transmissible cancer. Proc. Natl Acad.
Sci. USA 110, 9862–9867 (2013).
human obesity. antibiotics.

MILESTONES
example, the high-fibre diets that are

M I L E S TO N E 1 2 typically consumed by rural communities
were associated with increased microbial
Impact of diet–microbiota interactions diversity, an enrichment of Prevotella
species, higher concentrations of
on human metabolism health-promoting short-chain fatty acids
and a reduction in metabolic disease.
Studies on a range of other metabolic
diseases in relation to the microbiota
and diet, such as type 2 diabetes and
cardiovascular disease, also emerged.
A clear link between the ability of the
gut microbiota to metabolise dietary
phosphatidylcholine into trimethyl-
amine-N-oxide and the development of
cardiovascular disease was confirmed in
a series of papers by Stanley Hazen and
colleagues.
Given the substantial impact of diet
on the microbiota, numerous research
groups have attempted to harness
this power in order to modulate the
gut microbiota to alleviate metabolic
disease. In 2015, Zeevi et al. used gut
microbiota data, together with blood
parameters and metadata, to develop
a machine-learning algorithm that
could predict an individual’s glycemic
response to a particular meal, resulting
While studies had made it clear that identified no trend, the opposite trend in a personalized diet that could lower
our gut microbiota could metabolize or found that diet was in fact the main post-meal glucose. A more general
dietary components (MILESTONE 10), driver, rather than the obese state. These studies dietary intervention was recently used for
it was yet to be confirmed whether Given the conflicting results, three the treatment of type 2 diabetes mellitus.
these diet–microbiota interactions had reanalysis papers were published, using
highlight the In 2018, Zhao et al. used a high-fibre diet
implications for human health. publicly available datasets, in an attempt crucial impact to promote colonization by short-chain
Jeffrey Gordon’s group jump started to uncover conserved microbiota that diet can fatty acid producers and to improve
research into the links between the gut signatures of obesity. The overarching haemoglobin A1c levels, which was used
microbiota and obesity with a series of results were that phylum-level signatures
have on the as a readout of type 2 diabetes status.
mouse studies. In 2004, this group found were not generalizable, especially at gut microbiota These studies highlight the crucial
that germ-free mice had reduced body the population level, however Shannon and host impact that diet can have on the gut
fat compared to conventional mice, diversity and evenness, the number of microbiota and host metabolism, the
even though they consumed less food. operational taxonomic units, and obesity
metabolism resulting implications for human health,
A year later it was shown that a mouse status did have significant associations, and how we can use our knowledge
model of obesity had an altered ratio of albeit relatively weak. of these interactions to develop
the two main phyla present in the gut; However, diet was found to nutrition-based treatments.
the Bacteroidetes and the Firmicutes. A consistently alter the gut microbiota. For Emily White, Nature Microbiology
functional analysis of these microbiomes
revealed that an obesity-associated
ORIGINAL ARTICLE Ley, R. E. et al. Human gut microbes associated Le Chatelier, E. et al. Richness of human gut microbiome correlates
microbiota had an increased capacity with obesity. Nature 444, 1022–1023 (2006). with metabolic markers. Nature 500, 541–546 (2013) | Smith, M. I. et al.
for energy harvest, and this phenotype FURTHER READING Bäckhed, F. et al. The gut microbiota as an Gut microbiomes of Malawian twin pairs discordant for kwashiorkor.
could be transferred through faecal environmental factor that regulates fat storage. Proc. Natl Acad. Sci. Science 339, 548–554 (2013) | Tang, W. H. et al. Intestinal microbial
USA 101, 15718–157123 (2004) | Ley, R. E. et al. Obesity alters gut metabolism of phosphatidylcholine and cardiovascular risk. N. Engl.
microbiota transplant (MILESTONE 11). microbial ecology. Proc. Natl Acad. Sci. USA 102, 11070 (2005) | Cani, P. J. Med. 368, 1575–1584 (2013) | David, L. et al. Diet rapidly and
Members of the Gordon lab continued D. et al. Metabolic endotoxemia initiates obesity and insulin resistance. reproducibly alters the human gut microbiome. Nature 505, 559–563
this line of research, but this time in Diabetes 56, 1761–1772 (2007) | Turnbaugh, P. J. et al. A core gut (2014) | Subramanian, S. et al. Persistent gut microbiota immaturity in
microbiome in obese and lean twins. Nature 457, 480–484 (2009) | malnourished Bangladeshi children. Nature 510, 417–421 (2014) |
humans. In 2006, Ley et al. found that Hildebrandt, M. A. et al. High-fat diet determines the composition of Zeevi, D. et al. Personalized nutrition by prediction of glycemic
obese individuals had a reduction in the the murine gut microbiome independently of obesity. Gastroenterol. responses. Cell 163, 1079–1094 (2015) | Thaiss, C. A. et al. Persistent
relative abundance of Bacteroidetes 137, 1716–1724 (2009) | De Filippo, C. et al. Impact of diet in shaping microbiome alterations modulate the rate of post-dieting weight
gut microbiota revealed by a comparative study in children from regain. Nature 540, 544–551 (2016) | Zhao, L. et. al. Gut bacteria
compared to lean individuals, and
Europe and rural Africa. Proc. Natl Acad. Sci. USA 107, 14691–14696 selectively promoted by dietary fibers alleviate type 2 diabetes.
that this could be reversed using (2010) | Wang, Z. et al. Gut flora metabolism of phosphatidylcholine Science 359, 1151–1156 (2018) | Sze, M. A. & Schloss, P. D. Looking for a
diet. This triggered numerous studies promotes cardiovascular disease. Nature 472, 57–63 (2011) | Wu, G. D. signal in the noise: revisiting obesity and the microbiome. mBio 7,
et al. Linking long-term dietary patterns with gut microbial enterotypes. e01018-16 (2016) | Walters, W. A., Xu, Z. & Knight, R. Meta-analyses of
of the microbiota in the context of
Science 334, 105–108 (2011) | Ridaura, V. K. et al. Gut microbiota from human gut microbes associated with obesity and IBD. FEBS Lett. 588,
obesity and malnutrition. While many twins discordant for obesity modulate metabolism in mice. Science 4223–4233 (2014) | Finucane, M. M., Sharpton, T. J., Laurent, T. J. &
were in agreement and identified an 341, 1241214 (2013) | Cotillard, A. et al. Dietary intervention impact Pollard, K. S. A taxonomic signature of obesity in the microbiome?
obesity-associated microbiota, others on gut microbial gene richness. Nature 500, 585–588 (2013) | Getting to the guts of the matter. PLoS ONE 9, e84689 (2014).

MILESTONES
Along with his colleagues, most

Credit: Science Photo Library / Alamy Stock Photo
notably Sarah Dath, Gratia observed

antagonism between co-cultures of
different strains of E. coli. This effect
was attributed to a secreted factor,
which came to be known as ‘colicin’.
This protein now represents the first
described member of an unrelated
family of narrow-spectrum, bacteri-
ally-produced antibiotics known as
‘bacteriocins’.
Another important step in
understanding the role played by
the host’s microbiota in resistance to
enteropathogenic bacteria was made
in the 1950s, by Marjorie Bohnhoff
and colleagues at the University
of Chicago, and subsequently in
the early 1970s by Dirk van Waaij
and colleagues in the Netherlands.
Secondary infections are a common
occurrence following a course of
antibiotics, suggesting that some
M I L E S TO N E 1 3 perturbation of the microbiota
might be responsible. In order to
Mechanisms of
model this phenomenon, these
studies found that mice that had
their microbiota heavily-depleted
colonization resistance
by antibiotics were drastically more
susceptible to oral challenge with
even mildly pathogenic strains of
Salmonella or E. coli. A 1971 paper
In 1917, as war was tearing its unique E. coli strain is still used to by van Waaij and colleagues was
way across Europe, a fascinating this day as the active component of especially important for its coining
scientific observation was being The host’s the probiotic Mutaflor. of the term ‘colonization resistance’
made. The German physician microbiota In many ways, the findings of and placing it into a quantitative
Alfred Nissle had been looking for Nissle were built on earlier concepts framework.
can manifest
novel therapies to tackle enteric articulated by the ‘father’ of cellular The host’s microbiota can
infections, which in this pre-anti- colonization immunology Élie Metchnikoff, manifest colonization resistance
biotic era represented an enormous resistance who in a monograph in 1910 had through a number of potential
burden on troops. He noted that through a lauded the consumption of soured mechanisms, for example, ‘passively’
one soldier in particular, who had milk (rich in bacteria) as a means by out-competing bacteria for
participated in a military campaign
number of to stave-off infectious disease and space and trophic resources, or
in the Balkans, proved stubbornly potential enhance human longevity. Indeed, more actively by the generation of
resistant to dysentery when many mechanisms peasants from the Balkans and bacteriocidal factors. Three key
of his comrades had been laid low Caucasus had long-been famous papers in 2007 illuminated different
by the disease. Speculating that a not only for their centenarians but aspects of colonization resistance.
component of this soldier’s intestinal also for millennia-old traditions of The probiotic strain Lactobacillus
microbiota might be responsible yoghurt-making. However, while salivarius UCC118 is known to pro-
for this resistance, Nissle acquired it seemed that certain strains of duce the bacteriocin Apb118. Conor
stool samples and was able to isolate bacteria could have beneficial prop- Gahan and colleagues observed that
a strain of bacteria that came to be erties on their host, perhaps in part this probiotic strain could protect
known as Escherichia coli Nissle through their direct antagonism of mice against infection with Listeria
1917. Laboratory testing, as well as enteric pathogens, the mechanistic monocytogenes and this effect was
some self-experimentation on the basis of these remarkable effects wholly dependent on the production
part of Nissle, showed that this novel were almost wholly unknown. of Apb118. However, L. salivarius
strain of E. coli was indeed able to Arguably, the first in-roads into UCC118 also conferred protection
antagonise pathogenic bacteria and this question were made in the against a strain of Salmonella
it soon entered clinical practice. mid-1920s, in Belgium, by an resistant to Apb118, suggesting
Although his identity is lost to often-overlooked early pioneer that colonization resistance by this
history, the soldier’s donation of his of microbiology — André Gratia. probiotic is more multi-faceted than

MILESTONES
simply the production of a bacte- by another through the generation from the study of colonization resist-
riocin. A second pair of unrelated of biologically active factors. ance could offer the hope of novel
papers set out to understand how As we teeter towards the dangers antimicrobial therapies.
enteropathogens could overcome a post-antibiotic era, further insights Zoltan Fehervari, Nature Immunology
colonization resistance. In separate
mouse studies and using different ORIGINAL ARTICLES Corr, S. et al. Bacteriocin Proc. Natl Acad. Sci. USA 108, 17480–17485 (2011) |
enteropathogens (Citrobacter roden- production as a mechanism for the anti-infective Fukuda, S. et al. Bifidobacteria can protect from
tium or Salmonella), it was shown activity of Lactobacillus salivarius UCC118. Proc. Natl enteropathogenic infection through production of
Acad. Sci. USA 104, 7617–7621 (2007) | Lupp, C. et al. acetate. Nature 469, 543–547 (2011) | Kamada, N.
that intestinal inflammation altered Host-mediated inflammation disrupts the intestinal et al. Regulated virulence controls the ability of a
the composition of the host’s micro- microbiota and promotes the overgrowth of pathogen to compete with the gut microbiota.
Enterobacteriaceae. Cell Host Microbe 16, 119–129 Science 336, 1325–1329 (2012) | Buffie, C. G. et al.
biota and made them susceptible to
(2007) | Stecher, B. et al. Salmonella enterica serovar Precision microbiome reconstitution restores bile
colonization by the invading bacte- Typhimurium exploits inflammation to compete with acid mediated resistance to Clostridium difficile.
ria. In both cases the bacteria needed the intestinal microbiota. PLoS Biol. 5, 2177–2189 Nature 517, 205–208 (2015) | Sassone-Corsi, M. et al.
(2007). Microcins mediate competition among
to be able to elicit gut inflammation FURTHER READING Bohnhoff, M. et al. Effect of Enterobacteriaceae in the inflamed gut. Nature 540,
in order to establish themselves — in streptomycin on susceptibility of intestinal tract to 280–283 (2016) | Rivera-Chávez, F. et al. Depletion of
other words, this appeared to be a experimental Salmonella infection. Proc. Soc. Exp. butyrate-producing Clostridia from the gut
Biol. Med. 86, 132–137 (1954) | van der Waaij, D. et al. microbiota drives an aerobic luminal expansion of
case of the enteropathogen co-opting Colonization resistance of the digestive tract in Salmonella. Cell Host Microbe 19, 443–454 (2016) |
the host’s immune response to its conventional and antibiotic-treated mice. J. Hyg. 69, Faber, F. et al. Host-mediated sugar oxidation
405–411 | Freter, R. Experimental enteric Shigella and promotes post-antibiotic pathogen expansion.
advantage.
Vibrio infections in mice and guinea pigs. J. Exp. Med. Nature 534, 697–699 (2016) | Byndloss, M. X. et al.
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Credit: Antlii / Alamy Stock Photo

Functional human microbiota

analyses in vivo using ’omics
technologies
Eline Klaassens and colleagues applied a
metaproteomics approach to uncultured faecal
microbiota, providing the first insights beyond
taxonomic identification. This was followed by
numerous studies using ’omics methods, such as
metabolomics and metatranscriptomics, as well as
the development of multi-omics pipelines; methods
that are still uncovering the functions of the
microbiota today.
ORIGINAL ARTICLE Klaassens, E. S., de Vos, W. M. & Vaughan, metabolic biomarkers of Crohn’s disease. PLoS ONE 4, e6386 Molecular cartography of the human skin surface in 3D. Proc.
E. E. Metaproteomics approach to study the functionality of the (2009) | Martin, F. P. et al. Topographical variation in murine Natl Acad. Sci. USA 112, 2120–2129 (2015) | Heintz-Buschart, A.
microbiota in the human infant gastrointestinal tract. Appl. intestinal metabolic profiles in relation to microbiome et al. Integrated multi-omics of the human gut microbiome in a
Environ. Microbiol. 73, 1388–1392 (2007). speciation and functional ecological activity. J. Proteome Res. 8, case study of familial type 1 diabetes. Nat. Microbiol. 2, 16180
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metaproteomics of the human distal gut microbiota. ISME J. 3, metatranscriptome and metagenome of the human gut. Proc. metabolic activity in inflammatory bowel disease.
179–189 (2008) | Jansson, J. et al. Metabolomics reveals Natl Acad. Sci. USA 111, 2329–2338 (2014) | Bouslimani, A. et al. Nat. Microbiol. 4, 293–305 (2019).

MILESTONES
Antibiotics alter the gut microbiome and host health

Antibiotics not only act on bacteria asthma mouse model. The changes in
that cause infections but also affect community composition were accom-
the resident microbiota. Although panied by changes in metabolites such
this side effect has long been appreci- as acetate, a short chain fatty acid that
ated, advances in sequencing technol- is known to influence host metabo-
ogies enabled detailed study of how lism and immune function. In another
antibiotics alter the gut microbiome. study, early life exposure to the anti-
Although the composition of biotic vancomycin indeed increased
the gut microbiota varies between immunoglobulin E and decreased
individuals, the community in each regulatory T cell levels in mice.
individual is relatively stable over A study looking at antibiotic
time (MILESTONE 7). In 2008, Relman perturbation of the gut microbiota
and colleagues studied three healthy and the risk of developing inflam-
individuals and showed that treatment matory bowel disease, also showed
with ciprofloxacin influenced the that transfer of the disturbed gut
Credit: yulia Petrova / Alamy Stock Photo
abundance of about one third of microbiota from mouse mothers to
bacterial taxa in faecal samples. These their newborn pups, promoted and
changes decreased the taxonomic antibiotic treatment increased the accelerated the development of gut
richness, diversity and evenness of the incidence of type-1 diabetes in sus- inflammation in the offspring.
community. Although most bacterial Treatment with ceptible mice, and the relative levels Certain interventions could help
groups recovered after treatment, of anti-inflammatory T cells were restore the gut microbial community
ciprofloxacin
several taxa did not (even after six lower in these mice (prior to the onset to its original state if antibiotics are
months) and the level of reconstitu- influenced the of disease) than in untreated mice. needed. For example, Elinav and
tion varied between the individuals. A abundance Neonatal treatment with antibiotics colleagues recently found that the
follow-up study showed that a second of about increased the susceptibility of adult administration of certain probiotics
course of ciprofloxacin had similar mice to imiquimod-induced psoriasis. hindered, and autologous faecal
effects. There was no correlation
one third Similar observations have been transplantation helped restore, the
between the magnitude of the micro- of bacterial made for asthma. Children who are microbiome.
biome shift in the first and second taxa [and] exposed to antibiotics in their first The observation that the gut
treatments within any individual; each decreased the year of life had a slightly increased microbiome can be permanently
treatment was another ‘roll of the dice’. risk of developing asthma, and the perturbed even by short-term or
Owing to the close links between taxonomic risk increased with the number of low-dose antibiotic treatment, and
the resident microbiota and the richness, antibiotic courses. Regression analysis that this change can have long-term
host, such disturbance of the diversity and from children with asthma identified effects on health, cautions against
microbiota by antibiotics can be that early exposure to antibiotics is widespread and potentially unneces-
evenness of
expected to affect host physiology a risk factor. The abundance of the sary use of antibiotics, particularly in
and potentially host health. Indeed, the community genera Faecalibacterium, Lachnospira, young children and pregnant women,
one study found that administra- Veillonella and Rothia (FLVR) were and illustrates that antibiotics should
tion of antibiotics to young mice decreased in children at three months not be considered harmless. However,
increased adiposity and levels of of age who had been treated with anti- it also raises hopes for microbiome
metabolic hormones in the blood, biotics and later developed asthma. modulation as a therapeutic treatment
and faecal transplantation from In faecal transfer experiments from for immune conditions.
antibiotic-treated mice to germ-free one of these children, the addition of Megan Cully,
mice transferred the metabolic FLVR decreased disease severity in an Nature Reviews Drug Discovery
phenotype. Besides effects on
metabolism, the gut microbiota also ORIGINAL ARTICLES Dethlefsen, L. et al. The pervasive effects of an metabolic alterations affect risk of childhood asthma. Sci. Transl. Med.
closely interacts with and influences antibiotic on the human gut microbiota, as revealed by deep 16S rRNA 7, 307ra152 (2015) | Russell, S. L. et al. Perinatal antibiotic treatment
sequencing. PLoS Biol. 6, e280 (2008) | Dethlefsen, L, & Relman, D. A. affects murine microbiota, immune responses and allergic asthma. Gut
the host immune system, and thus Incomplete recovery and individualized responses of the human distal Microbes 4, 158–164 (2013) | Livanos, A. E. et al. Antibiotic-mediated
microbiome disturbances have the gut microbiota to repeated antibiotic perturbation. Proc. Natl Acad. gut microbiome perturbation accelerates development of type 1
potential to affect the development of Sci. USA 108, 4554–4561 (2011) | Cho, I. et al. Antibiotics in early life diabetes in mice. Nat. Microbiol. 1, 16140 (2016) | Schulfer, A.F. et al.
alter the murine colonic microbiome and adiposity. Nature 488, Intergenerational transfer of antibiotic-perturbed microbiota
several autoimmune, inflammatory 621–626 (2012) | Cox, L. M. et al. Altering the intestinal microbiota enhances colitis in susceptible mice. Nat. Microbiol. 3, 234–242 (2018) |
and allergic diseases. during a critical developmental window has lasting metabolic Suez, J. et al. Post-antibiotic gut mucosal microbiome reconstitution is
consequences. Cell 158, 705–721 (2014) | Zanvit, P. N. et al. Antibiotics impaired by probiotics and improved by autologous FMT. Cell 174,
Antibiotic treatment of mice from in neonatal life increase murine susceptibility to experimental 1406–1423 (2018)
birth also alters the expression of psoriasis. Nat. Commun. 6, 8424 (2015) | Marra, F. et al. Antibiotic use in FURTHER READING Willing, C. P. et al. Shifting the balance: antibiotic
genes and the number of cells that children is associated with increased risk of asthma. Pediatrics 123, effects on host-microbiota mutualism. Nat. Rev. Microbiol. 9, 233–243
1003–1010 (2009) | Arrieta, M. C. et al. Early infancy microbial and (2011)
regulate immune responses. Pulsed

MILESTONES
Bioinformatics
tools facilitate
the analysis of
microbiome
sequencing data
The software pipeline QIIME, which stands for
Credit: N. Smith / Springer Nature Limited
‘quantitative insights into microbial ecology’,

enables the analysis and interpretation of
the increasingly large datasets generated by
microbiome sequencing.
ORIGINAL ARTICLE Caporaso, J. G. et al. QIIME allows analysis

of high-throughput community sequencing data. Nat. Methods
7, 335–336 (2010).
FURTHER READING Knight, R. et al. Best practices for
analysing microbiomes. Nat. Rev. Microbiol. 16, 410–422 (2018).
CALL FOR PAPERS
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MILESTONES
Furthermore, it has been possible to
Credit: Panther Media GmbH / Alamy Stock Photo

identify disease-specific microbiome
signals, for example, for type-1 diabe-
tes mellitus (MILESTONE 9), metabolic
syndrome, obesity (MILESTONE 12),
inflammatory bowel disease and oth-
ers. Notably, the second phase of the
Human Microbiome Project used an
integrative approach that combines
several ’omics techniques to study the
role of the microbiome in preterm
birth, the development of type-2
diabetes mellitus and inflammatory
bowel disease over time. Identifying
such signals is a first step towards
understanding how the microbiome
might contribute to disease develop-
ment, and towards the development
of preventative and therapeutic
applications.
Microbiome analyses in large For example, individual microbi-
ome differences are associated with
human populations the response to cancer treatment

(MILESTONE 24). This finding and
other studies have inspired plans
Microbiome composition and func- by contrast, today’s studies can con- to exploit microbiome differences
tion have been implicated in various tain samples from several thousand for individualized therapies and
diseases. However, understanding participants. Larger numbers, as well Besides the interventions.
and exploiting the interactions of the as refinement and standardisation In summary, large population
microbiome with the human host of protocols and pipelines, and the
scientific studies have greatly advanced our
is tempered by the huge diversity of availability of larger reference data achievement understanding of gut microbiome
the microbiome within and between sets — all of which have profited […], one should diversity and have identified numerous
individuals. Advances in metagenom- hugely from early microbiome note and potential links to health and disease,
ics and high-throughput sequencing population studies — add to the inspiring many new research avenues.
in the early 2000s, inspired projects robustness of results. Sometimes it acknowledge They have also made essential con-
aimed at capturing microbiome has been difficult to compare studies the fruitful tributions to establishing methods
diversity in large human populations. and there have been studies with collaboration and standards on which future work
For example, one of the first such pro- contradictory results. Nevertheless, can build. Finally, these studies have
of these large
jects, Metagenomics of the Human large population studies have greatly highlighted the importance of the
Intestinal Tract, studied faecal sam- advanced our understanding of scientific microbiome, not only for scientists
ples from 124 individuals, generating what constitutes a ‘normal’ human consortia but also for the general public.
576.7 gigabases of sequence data. microbiome, although this statement Ursula Hofer,
The authors noted that this is “almost should be qualified by the fact that Nature Reviews Microbiology
200 times more than in all previous North Americans and Europeans are
studies” and “provides a broad view of the best-studied populations. Projects ORIGINAL ARTICLE Qin, J. N. et al. A human gut microbial gene catalogue
the functions important for bacterial are underway to ameliorate this bias. established by metagenomic sequencing. Nature 464, 59–65 (2010)
life in the gut”. In general, the microbiome differs FURTHER READING The Human Microbiome Project Consortium. Structure, function
and diversity of the healthy human microbiome. Nature 486, 207–214 (2012) | The
Besides the scientific achievement not only between healthy individ- Human Microbiome Project Consortium. A framework for human microbiome research.
of this and similar projects, such as uals and those with diseases, even Nature 486, 215–221 (2012) | Goodrich, J. K. et al. Human genetics shape the gut
microbiome. Cell 159, 789–799 (2014) | Ding, T. & Schloss, P. D. Dynamics and
the Human Microbiome Project, between healthy people there is a associations of microbial community types across the human body. Nature 509, 357–360
Belgian Flemish Gut Flora Project, large diversity — there is no uniform (2014) | Falony, G. et al. Population-level analysis of gut microbiome variation. Science
Dutch LifeLines-DEEP study and ‘healthy’ microbiome. Some general 352, 560–564 (2016) | Zhernakova, A. et al. Population-based metagenomics analysis
reveals markers for gut microbiome composition and diversity. Science 352, 365–369
others, one should note and acknowl- measures, such as high taxonomic (2016) | Rothschild, D. et al. Environment dominates over host genetics in shaping
edge the fruitful collaboration of and functional richness, which human gut microbiota. Nature 555, 210–215 (2018) | McDonald, D. et al. Amercian gut:
these large scientific consortia, lately usually correlate with a diverse, fibre- an open platform for citizen science microbiome research. mSystems 3, e00031-18
(2018) | He, Y. et al. Regional variation limits applications of healthy gut microbiome
even including citizen scientists in rich diet, seem to be beneficial. references and disease models. Nat. Med. 24, 1532–1535 (2018). | Fettweis, J. M. et al. The
American Gut. Furthermore, dedi- Population studies have also vaginal microbiome and preterm birth. Nat. Med. https://doi.org/10.1038/s41591-019-
0450-2 (2019) | Zhou, W. et al. Complex host-microbial dynamics in prediabetes revealed
cated support from several funders identified factors that shape the through longitudinal multi-omics profiling. Nature 569, 663–671 (2019) | Lloyd-Price, J.
has been essential. microbiome, and have helped quan- et al. Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature
As mentioned above, initial stud- tify their impact, including body site, 569, 655–662 (2019) | Integrative HMP (iHMP) Research Network Consortium. The
Integrative Human Microbiome Project. Nature 569, 641–648 (2019)
ies looked at around 100 individuals; diet, drugs, host genetics and others.

MILESTONES
M I L E S TO N E 1 8 Credit: K. Lee / Springer Nature Limited
The microbiota–gut–brain axis

A link between the gut microbiota and the brain interactions. Spore-forming gut bacteria were
has long been surmised, but in recent decades, found to drive the production of serotonin by
studies have started to report causal effects of enterochromaffin cells in the mouse colon,
the gut microbiota on our brains and behaviour, although exactly how this may affect the brain has
and the underlying molecular mechanisms have not been clear. Moreover, male (but not female) GF
begun to be elucidated. mice show higher levels of hippocampal serotonin
Several early studies in animal models provided and plasma levels of a serotonin precursor,
evidence that stress can perturb the composition suggesting that certain influences of the gut
of the gut microbiota and that enteric pathogens microbiota on the brain may be sex-specific.
can affect host behaviour. In 2004, a study showed How the gut microbiota signal to the brain has
that germ-free (GF) mice exhibit an upregulated been the focus of much research. Evidence from
hormonal response to stress induced by physical models of multiple sclerosis and stroke suggested
restraint, implying that the microbiota influences that changes in the gut microbiota may indirectly
the neuroendocrine hypothalamic–pituitary– influence the central nervous system via effects disorder associated with α-synuclein
adrenal (HPA) axis, the central stress response on immune homeostasis and immune responses. aggregation in the brain) the presence of gut
system. However, the effects of the microbiota In support of a vagus-nerve mediated route for microbiota or microbially produced short-chain
— or the absence thereof — on behaviour gut-derived signals, severing the vagus nerve fatty acids promoted neuroinflammation, motor
remained unclear. Seven years later, in 2011, below the diaphragm blocked the anxiolytic and impairments and α-synuclein pathology.
several experimental findings in mice shed light gene expression effects of L. rhamnosus (JB-1). By Nearly all of the work in this field to date
on how a lack of conventional microbiota affects contrast, ablating the vagus nerve or sympathetic has been carried out in animal models, and
behaviour, gene expression in the brain and the nerves did not prevent the effects of ATM on establishing whether those findings translate
development of the nervous system. anxiety-like behaviour, and ATM-treated mice to humans will be crucial yet challenging. As
Studies revealed that GF and antibiotic-treated showed no overt signs of gut inflammation or an example of such an endeavour, a study
mice displayed reduced anxiety-like behaviour alterations in enteric neurotransmitter levels, investigated the link between faecal microbiota
compared with specific pathogen-free (SPF) indicating that some gut–brain communication composition and quality of life using data from
controls. For example, GF mice were found routes might be independent of the immune and more than 1,000 people. As well as identifying
to spend more time on the open arms of the nervous systems. bacterial genera associated with higher
elevated plus maze (EPM), and in the illuminated In fact, later research has started to uncover quality of life or depression, they carried out
compartment of the light–dark box, than their other means of gut–brain communication — in metagenomic analyses that indicated that
SPF counterparts. The offspring of GF mice that particular, microorganism-derived products the potential of microorganisms to synthesize
had been conventionalized with SPF microbiota, that can directly or indirectly signal to the certain neuroactive metabolites may also
but not GF mice conventionalized as adults, nervous system. For example, the offspring of correlate with mental wellbeing.
showed behaviour similar to SPF controls, immune-challenged mice showed gut dysbiosis, Together, the studies described above have
suggesting that the microbiota may influence the disrupted intestinal integrity and behavioural laid the foundations for our understanding of the
brain during a ‘critical period’ of development. abnormalities (including anxiety-like behaviour), effects of the gut microbiota on the brain and
Related work showed an effect of differences as well as high serum levels of a microbial behaviour, and the mechanisms that underlie
in gut microbiota on behaviour. Mice treated metabolite that, when injected into wild-type them, and represent initial efforts to explore the
with a mixture of antimicrobials (ATM) showed mice, induced anxiety-like behaviour. Similarly, relevance of animal-model findings for humans.
more exploratory behaviour, and GF BALB/c in a model of Parkinson disease (a neurological Natasha Bray, Nature Reviews Neuroscience
mice (which are typically timid) colonized with
microbiota from another mouse strain exhibited
ORIGINAL ARTICLES Diaz Heijtz, R. et al. Normal gut Sampson, T. R. et al. Gut microbiota regulate motor deficits and
more exploratory behaviour than those receiving microbiota modulates brain development and behavior. Proc. neuroinflammation in a model of Parkinson’s disease. Cell 167,
BALB/c microbiota, and vice versa. Furthermore, Natl Acad. Sci. USA 108, 3047–3052 (2011) | Neufeld, K. M. et al. 1469−1480 (2016) | Valles-Colomer, M. et al. The neuroactive
it was found that treatment of SPF mice with the Reduced anxiety-like behavior and central neurochemical potential of the human gut microbiota in quality of life and
change in germ-free mice. Neurogastroenterol. Motil. 23, depression. Nat. Microbiol. 4, 623–632 (2019) | Desbonnet, L. et al.
probiotic Lactobacillus rhamnosus (JB-1) reduced 255–264 (2011) | Bercik, P. et al. The intestinal microbiota affect Microbiota is essential for social development in the mouse.
anxiety- and depression-like behaviour. central levels of brain-derived neurotropic factor and behavior Mol. Psychiatry 19, 146–148 (2014) | De Vedder, F. et al.
As well as behavioural differences, the brains in mice. Gastroenterol. 141, 599–609 (2011) | Bravo, J. A. et al. Microbiota-generated metabolites promote metabolic benefits
Ingestion of Lactobacillus strain regulates emotional behavior via gut-brain neural circuits. Cell 156, 84–96 (2014) | Olson, C. A.
of animals with altered or absent gut microbiota and central GABA receptor expression in a mouse via the vagus et al. The gut microbiota mediates the anti-seizure effects of the
displayed various molecular differences. These nerve. Proc. Natl Acad. Sci. USA 108, 16050–16055 (2011). ketogenic diet. Cell 173, 1728–1741 (2018) | Buffington, S. A.
included brain-region-specific changes in FURTHER READING Sudo, Y. et al. Postnatal microbial et al. Microbial reconstitution reverses maternal diet-induced
colonization programs the hypothalamic–pituitary–adrenal social and synaptic deficits in offspring. Cell 165, 1762–1775
levels of brain-derived neurotrophic factor
system for stress response in mice. J. Physiol. 1, 263–275 (2004) | (2016) | Kim, S. et al. Maternal gut bacteria promote
(BDNF; which is known to be modulated in Yano, J. et al. Indigenous bacteria from the gut microbiota neurodevelopmental abnormalities in mouse offspring. Nature
anxiety and depression), differences in the regulate host serotonin biosynthesis. Cell 161, 264–276 (2015) | 549, 528–532 (2017) | Schretter, C. E. et al. A gut microbial factor
Clarke, G. et al. The microbiome-gut-brain axis during early life modulates locomotor behaviour in Drosophila. Nature 563,
expression of various neurotransmitter receptors
regulates the hippocampal serotonergic system in a sex- 402–406 (2018) | Ochoa-Repáraz, J. et al. Role of gut commensal
and alterations in the turnover of certain dependent manner. Mol. Psychiatry 18, 666–673 (2013) | microflora in the development of experimental autoimmune
neurotransmitters, including serotonin. Hsiao, E. Y. et al. Microbiota modulate behavioral and encephalomyelitis. J. Immunol. 183, 6041–6050 (2009) | Singh, V.
Indeed, much research since has focused on physiological abnormalities associated with et al. Microbiota dysbiosis controls the neuroinflammatory
neurodevelopmental disorders. Cell 155, 1451−1463 (2013) | response after stroke. J. Neurosci. 36, 7428–7440 (2016).
serotonin as a node of gut microbiota–brain

MILESTONES
Modern culturing
efforts expand the
culturable microbiota
Credit: Ridvan Arda / Alamy Stock Photo

High-throughput anaerobic culturing enabled the
recovery of a large part of the diverse human gut
microbiota and the creation of individual culture
collections.
ORIGINAL ARTICLE Goodman, A. L. et al. Extensive personal human

gut microbiota culture collections characterized and manipulated in
gnotobiotic mice. Proc. Natl Acad. Sci. USA 108, 6252–6257 (2011).
FURTHER READING Lagier, J. C. et al. Culturing the human microbiota
and culturomics. Nat. Rev. Microbiol. 1, 540–550 (2018).
Global human microbiome

Genetic variation occurs between human cohorts living in different regions, including
populations living in different places, the Amazonas of Venezuela, rural Malawi
but little was known about variation and US metropolitan areas. The authors
in microbiomes. To investigate how found pronounced differences in the
gut microbiomes differ among human composition and functions in the gut
Credit: N. Wallington / Springer Nature Limited
populations, Yatsunenko et al. characterized microbiomes between these geographically

bacterial species in faecal samples from distinct cohorts.
ORIGINAL ARTICLE Yatsunenko, T. et al. Human gut microbial and functional uniqueness. DNA Res. 23, 125–
microbiome viewed across age and geography. Nature 133 (2016) | Das, B. et al. Analysis of the gut microbiome
486, 222–227 (2012). of rural and urban healthy Indians living in sea level and
FURTHER READING Schnorr, S. L. et al. Gut microbiome high-altitude areas. Sci. Rep. 8, 10104 (2018) | Pasolli, E.
of the Hadza hunter-gatherers. Nat. Commun. 5, 3654 et al. Extensive unexplored human microbiome diversity
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African Americans and rural Africans. Nat. Commun. 6, spanning age, geography, and lifestyle. Cell 176, 649–662
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strategies in traditional societies distinguish gut Kyrpides, N. Novel insights from uncultivated genomes of
microbiomes. Nat. Commun. 6, 6505 (2015) | Nishijima, S. the global human gut microbiome. Nature https://doi.
et al. The gut microbiome of healthy Japanese and its org/10.1038/s41586-019-1058-x (2019).

Microbially-produced
short-chain fatty acids induce
regulatory T cell production
Regulatory T cells (Tregs) are crucial in maintenance ORIGINAL ARTICLES Smith, P.M. et al. The microbial T cells. Nat. Rev. Immunol. 16, 295–309 (2016) |
of immune homeostasis. In 2013, three studies metabolites, short-chain fatty acids, regulate colonic Round, J. L. & Mazmanian, S. K. Inducible
Treg cell homeostasis. Science 341, 569–573 (2013) | Foxp3+ regulatory T-cell development by a
found that microbiota-derived short-chain fatty Atarashi, K. et al. Treg induction by a rationally selected commensal bacterium of the intestinal microbiota.
acids promote the expansion and differentiation mixture of Clostridia strains from the human microbiota. Proc. Natl Acad. Sci. USA 107, 12204–12209 (2010) |
of Tregs, revealing a form of chemical-mediated Nature 500, 232–236 (2013) | Arpaia, N. et al. Metabolites Geuking, M. B. et al. Intestinal bacterial colonization
produced by commensal bacteria promote peripheral induces mutualistic regulatory T cell responses. Immunity
communication between the commensal microbiota regulatory T-cell generation. Nature 504, 451–455 (2013). 34, 794–806 (2011) | Lathrop, S. K. et al. Peripheral
and the immune system that affects immune FURTHER READING Tanoue, T., Atarashi, K. & Honda, K. education of the immune system by colonic commensal
mechanisms. Development and maintenance of intestinal regulatory microbiota. Nature 478, 250–254 (2011).

MILESTONES
M I L E S TO N E 2 2 M I L E S TO N E 2 3
Production Host-targeted drugs affect

of antibiotics microbiota populations
by the human Commonly used medications affect gastrointestinal microbial
abundances and bacterial gene expression, which may both positively
microbiota and negatively
Library / Alamy Stock Photo

contribute to the
Credit: Science Photo

Identification of biosynthetic effects on human
gene clusters for antibiotics health associated
ited
in the genomes of the with drug
Lim
human microbiota, treatment.
re
tu
suggests new sources of
Na
er
antimicrobial drugs whose g
rin
/ Sp
species-specific production y ORIGINAL ARTICLES Tsuda A et al. Influence of proton-pump inhibitors on the luminal microbiota
rsb
has the potential to modulate me in the gastrointestinal tract. Clin. Transl. Gastroenterol. 6, e89 (2015) | Freedberg, D. E. et al. Proton
Sum
Credit: V. pump inhibitors alter specific taxa in the human gastrointestinal microbiome: a crossover trial.
the local microbial community Gastroenterology 149, 883–885 (2015) | Forslund, K. et al. Disentangling type 2 diabetes and
structure. metformin treatment signatures in the human gut microbiota. Nature 528, 262–266 (2015).
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ORIGINAL ARTICLE Donia, M. S. et al. A systematic analysis of biosynthetic gene clusters in physiology and gene expression of the active human gut microbiome. Cell 152, 39–50 (2013) |
the human microbiome reveals a common family of antibiotics. Cell 158, 1402–1414 (2014). Maier L. et al. Extensive impact of non-antibiotic drugs on human gut bacteria. Nature 555,
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impair pathogen colonization. Nature 535, 511–516 (2016). pharmacokinetics and toxicity. Science 363, eaat9931 (2019).
Human microbiota affects response to cancer therapy

Following earlier studies in mouse models, gut microbiota composition was shown to affect the
response of melanoma patients, and those suffering from advanced lung or kidney cancer, to
immune checkpoint therapy, as well as tumour control.
ORIGINAL ARTICLES Routy, B. et al. Gut microbiome cancer response to therapy by modulating the tumor
influences efficacy of PD-1-based immunotherapy against microenvironment. Science 342, 967–970 (2013) | Viaud, S. et al.
epithelial tumors. Science 359, 91–97 (2018) | Gopalakrishnan, V. The intestinal microbiota modulates the anticancer immune
et al. Gut microbiome modulates response to anti-PD-1 effects of cyclophosphamide. Science 342, 971–976 (2013) | Taur, Y.
immunotherapy in melanoma patients. Science 359, 97–103 et al. The effects of intestinal tract bacterial diversity on mortality
Credit: ImageSource
(2018) | Matson, V. et al. The commensal microbiome is following allogeneic hematopoietic stem cell transplantation.
associated with anti-PD-1 efficacy in metastatic melanoma Blood 124, 1174–1182 (2014) | Sivan, A. et al. Commensal
patients. Science 359, 104–108 (2018). Bifidobacterium promotes antitumor immunity and facilitates
FURTHER READING Tanoue, T. et al. A defined commensal anti-PD-L1 efficacy. Science 350, 1084–1089 (2015) | Vétizou, M.
consortium elicits CD8 T cells and anti-cancer immunity. Nature et al. Anticancer immunotherapy by CTLA-4 blockade relies on
565, 600–605 (2019) | Iida, N. et al. Commensal bacteria control the gut microbiota. Science 350, 1079–1084 (2015).
Credit: N. Wallington / Springer Nature Limited
Metagenome-assembled genomes
provide unprecedented characterization
of human-associated microbiota
Advances in computational methods, bacterial species from the gut and other body ORIGINAL ARTICLES Pasolli, E. et al. Extensive unexplored human
microbiome diversity revealed by over 150,000 genomes from
recently pioneered in the environmental sites, of global populations from rural and metagenomes spanning age, geography, and lifestyle. Cell 176,
microbiology field, enable the reconstruction urban settings, substantially expanding the 649–662 (2019) | Almeida, A. et al. A new genomic blueprint of the
of bacterial genomes from metagenomic known phylogenetic diversity and improving human gut microbiota. Nature https://doi.org/10.1038/s41586-019-
0965-1 (2019) | Nayfach, S. et al. New insights from uncultivated
datasets. This approach was used to identify classification of understudied, non-Western genomes of the global human gut microbiome. Nature https://doi.
thousands of new uncultured candidate populations. org/10.1038/s41586-019-1058-x (2019).

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June 2019

Human microbiota research

Produced by: Nature, Nature Microbiology, With support from:

1944 Culturing anaerobes (MILESTONE 1)

1958 Faecal microbiota transplantation for Clostridioides difficile infection (MILESTONE 2)

1965 Gut microbiota transfer experiments in germ-free animals (MILESTONE 3)

1972 The microbiota influences metabolism of host-directed drugs (MILESTONE 4)

1981 Microbiota succession in early life (MILESTONE 5)

1996 Sequence-based identification of human associated microbiota (MILESTONE 6)

1998 Stability and individuality of adult microbiota (MILESTONE 7)

2003 Beyond bacteria: studies of other host-associated microorganisms (MILESTONE 8)

2004 Regulation of mucosal immunity by the microbiota (MILESTONE 9)

2005 The importance of adequately feeding your microbiota (MILESTONE 10)

2006 Transfer of host phenotypes through microbiota transplantation (MILESTONE 11)

2006 Impact of diet–microbiota interactions on human metabolism (MILESTONE 12)

2007 Mechanisms of colonization resistance (MILESTONE 13)

2010 Microbiome analyses in large human populations (MILESTONE 17)

2011 The microbiota–gut–brain axis (MILESTONE 18)

2012 Global human microbiome (MILESTONE 20)

Microbially-produced short-chain fatty acids induce regulatory T cell production

2014 Production of antibiotics by the human microbiota (MILESTONE 22)

2015 Host-targeted drugs affect microbiota populations (MILESTONE 23)

2018 Human microbiota affects response to cancer therapy (MILESTONE 24)

Metagenome-assembled genomes provide unprecedented characterization of

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NATURE MILESTONES | HUMAN MICROBIOTA RESEARCH JUNE 2019 | S3

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M I L E S TO N E 1 revealed that the majority of envi-

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M I L E S TO N E 2 community structure in the colon.

Faecal microbiota transplantation is recruiting patients to a clinical

for Clostridioides difficile infection C. difficile-associated disease. FMT

patient and clinician convenience,

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between different species of the

Gut microbiota transfer elucidating the close links between

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Credit: G. Marshall / Springer Nature Limited

The microbiota influences metabolism of

Microbiota succession in early life

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The environment and people that

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M I L E S TO N E 6 accurate and complete assembly of genome

Sequence-based identification in resolving long repetitive regions. Thus, ref-

volunteers, covering a number of anatomical

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M I L E S TO N E 7 from 576.6 gigabases of metagenomic sequences

Stability and individuality of

adult microbiota that examined the faecal microbiomes of six

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fungal community of humans) has also been

fruitfully studied. A number of recent studies

Beyond bacteria: studies of other studies, suggest a complex interplay between

host-associated microorganisms Recently, the human archaeome (the

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Credit: S. Bradbrook / Springer Nature Limited

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