The world within: living with our microbial guests
EDWARD N. JANOFF, CLAIRE GUSTAFSON, and DANIEL N. FRANK
AURORA AND DENVER, COLO
of microorganisms of varying origins. These microbes
include archaea, bacteria, viruses, bacteriophage, and
eukaryotes (uni- and multicellular parasites). Although
constituting a potential threat to health and well-being
through parasitism, these microbial communities have
co-evolved over millions of years with the healthy
human host to provide a range of beneficial, and often
essential, services. Although still rudimentary, our
understanding of the beneficial roles played by the
human microbiome has grown appreciably in recent
years, as high-throughput, culture-independent technol-
ogy and related molecular technologies have been adap-
ted to complement the role of traditional microbiologic
cultures to facilitate the study of the human ecosystem.
rom the moments after our birth1and throughout
our lives, humans serve as reservoirs for
extremely complex and dynamic communities
Most studies identify individuals within complex popu-
lations by focusing on molecular characterization of
DNA from bacterial 16S ribosomal genes, a sequence
that distinguishes each organism by its phylum, genus,
and even species, depending on the length of the se-
quence. By studying communities in their native habi-
tats, rather than in liquid broth or on Petri plates, we
have gained significant insight into the dynamic, multi-
factorial interactions that occur among host, pathogen,
commensalcommunity, and environment. Under
normal, healthy circumstances, these interactions occur
across both the integument and mucosal surfaces
(eg, airways, intestinal, reproductive tracts, mouth),
the surfaces of the human body exposed to the environ-
ment that are the primary sites of microbial residence.
As thoughtfully reviewed in this issue of Translational
Research, the authors consider the microbial ecology
of the intestine,2the lung,3and the female reproductive
tract,4each of which supports diverse bacterial commu-
nities and, more recently appreciated, viruses5that en-
gage the host on multiple levels. Indeed, even skin has
now been exposed to reveal an abundance of microbial
Streptococcal species we were taught to expect, and
with heretofore unanticipated effects on host response.9
Ofparticular relevance arethehostandenvironmental
bility of the microbiome (Table I). The microbiome oc-
cupies a unique ecological niche at each bodily site.
space’’ in which multiple factors coalesce to support or
limit the selection of its members and to demarcate its
boundaries. These factors can include temperature, hu-
tors, competition with and resistance to other microbes
and their secreted products, and the activity of passive
From the Mucosal and Vaccine Research Colorado, Division of
Infectious Diseases, Department of Medicine, University
Colorado Denver School of Medicine, Aurora, Colo; Denver
Veterans Affairs Medical Center, Denver, Colorado; Microbiome
Research Consortium, University of Colorado Denver School of
Medicine, Aurora, Colo.
This work was supported by the Mucosal and Vaccine Research Col-
orado Program; National Institutes of Health Grants R01HD059527,
R21AI083615, and R21HG005964; the Dean’s Strategic Research
Committee grant to Mucosal and Vaccine Research Colorado Pro-
gram; and the Veterans Affairs Research Service.
Submitted for publication May 17, 2012; accepted for publication
May 17, 2012.
Reprint requests: Edward N. Janoff, Mucosal and Vaccine Research
Colorado, University of Colorado Denver, 12700 E. 19th Ave, Box
B-168, Aurora, CO 80045; e-mail: Edward.Janoff@ucdenver.edu.
1931-5244/$ - see front matter
? 2012 Published by Mosby, Inc.
ter host-derived effects may be modulated by the mi-
crobes’ ability to upregulate or downregulate and to
evade or subvert host recognition and response. Under-
standing the determinants of the fitness of the micro-
biome could help us facilitate its restoration when
The microbiome at each site serves the needs of the
host as well as its own. For example, the trillions of mi-
crobial cells and hundreds of species that colonize the
intestine have evolved metabolic pathways capable of
extracting energy from mammalian dietary inputs.
Rather than drawing down the energy content that is
available for absorption by the intestine, the enteric mi-
crobiome, particularly members of the bacterial phyla
Firmicutes (ie, low G1C gram-positive organisms
such as Clostridia) and the gram-negative Bacteroi-
detes, transform otherwise indigestible material, such
as complex plant polysaccharides, into fermentation
products that are more readily metabolized by mam-
additional calories and thereby extend the human geno-
mic capacity to harvest energy from foodstuffs.
rich soil precludes invasion by dandelions and weeds, an
intact microbiome limits colonization and clinical infec-
tant, organisms. Antibiotics disrupt the integrity and
to Clostridium difficile, enterococci, methicillin-resistant
The predisposition to infection conferred by antibiotics
may well extend beyond interrupting the competitive
inhibition of the pathogens by the normal commensal
species to include their effects on the epithelium,
metabolism, regulation of inflammasomes,11,12
fluenza, antibacterial treatment significantly limited their
ability to generate specific antiviral antibodies, CD41
and CD81 T-cell and interferon-a responses, and to con-
trol viral replication compared with those in untreated
control animals,14suggesting a role for bacteria in gener-
ating immune responses.
One of the most intriguing aspects of our understand-
ing of the role and regulation of the microbiome is its
interaction with the immune system. In animal models,
the acquisition of intestinal microbiota drives immune
development and maturation from birth, but mainte-
nance of intestinal homeostasis between immune com-
petence and tolerance is also critical to the proper
control of inflammation and progression to disease
states. These interactions are subserved by the juxtapo-
ically distinct areas, such as the surface epithelium,
inductive sites (eg, isolated or aggregated germinal cen-
ters), and the more diffuse effector sites in the lamina
propria (Fig 1). Initial immune interactions with
microbes are mediated by innate immune surface and
intracellular pattern recognition receptors, such as
toll-like receptors, NOD-like receptors, and retinoic
ficiency of the toll-like receptor 5 gene, which encodes
with a change in gut microbiota and significant
Table I. Definitions
Microbe: Any microscopic life form. Commonly bacteria or archaea, but many eukaryotes are microbes.
Microbiome: An assemblage of microbes in a particular time and place.
Virome: An assemblage of viruses in a particular time and place.
Microbial community: An assemblage of functionally and metabolically interacting microbes.
Metagenome: A composite genome from all organisms in a microbiome.
Meta-transcriptome: A composite gene expression profile of all organisms in a microbiome.
Richness: The number of bacterial species present in a population (alpha diversity, measured by Good’s coverage).
Diversity: The ‘‘complexity’’ or relative distribution of different species present in a population, ecosystem, or biome (beta diversity, measured
by Morisita-Horn Index). The similarities between distributions can also be determined.
Phylotype: A group of individuals characterized by their phylogenetic relationship to each other; a statistically associated shared richness and
diversity of specific organisms within an anatomic site, initially based on an evolutionary relationship.
Dysbiosis: Disease-associated alteration in the composition of a microbial community.
Pathobiont: A member of the microbiota, often antimicrobial-resistant, that can cause disease on perturbation of the otherwise constraining
Probiotics:Live commensalmicrobialorganisms (eg,LactobacillusGG,lactobacilli,bifidobacteria,Streptococci,or Saccharomycesboulardii,
alone or in combination) administered to enhance or suppress mucosal integrity, inflammation, or immune response.
Common Bacterial Phyla:
Bacteroides: Obligately anaerobic gram-negative bacteria. Prevalent commensals in human gut (eg, Bacteroides fragilis).
Firmicutes: Very diverse phylum of low G-C gram-positive bacteria, including staphylococci, streptococci, bacillii, and clostridia. Prevalent
commensals in human gut.
Proteobacteria: Very diverse phylum of gram-negative bacteria, including enterobacteriaceae (eg, Escherichia coli).
Actinobacteria: High G-C gram-positive bacteria, including mycobacteria and corynebacteria.
Janoff et al
metabolic perturbations inthe murine host, changes that
can conveyed to a wild-type host by transfer of stool.15
Commensal bacteria can elicit selectiveimmunologic
effects (Fig 1). In the mouse intestine, segmented fila-
mentous bacteria induce inflammatory T-helper 17 cells
that protect against bacterial and fungal infections.16
Conversely, Bacteroides fragilis and members of the
Clostridium groups IV and XIVa induce development
Fig 1. Specificcolonizingbacteria elicit innate immuneresponsesand development ofT cells and IgA-producing
cells in the intestine. (1) Bacteria colonize the lumen and interact with epithelial cells. Bacteria can be transported
directly through the epithelium by Microfold cells. Binding and uptake of bacteria or their products by epithelial
cells can activate innate receptors (toll-like receptor, NOD, retinoic acid-inducible gene-1–like receptors) and
stimulate cytokine secretion from the basolateral surface. DCs extending through the epithelium can sample an-
enhance differentiation of Tregand segmented filamentous bacteria in the development of T-helper 17 cells. (2) In
the lymphoid follicle or germinal center, na€ ıve IgD1IgM1B cells are activated by bacterial antigens. In associ-
ation with epithelial-derived soluble factors, these cells are committed to undergo class switch recombination to
IgA and somatic hypermutation under the influence of DCs, follicular helper CD41 T cells (T follicular helper
cells), and T-helper cells. These committed B cells then leave the follicle, transit through the lymph and blood,
and return or ‘‘home’’ predominantly to the lamina propria effector sites from which theyoriginated. With support
from Tregand T-helper cells, the returning B cells in the lamina propria differentiate into IgA-producing plasma
cells. (3) The polymeric IgA produced binds to polymeric IgA receptors on the basolateral surface of epithelial
cells and is transported into the lumen to bind bacteria and their antigens to limit adherence to, activation of,
and transport through epithelial cells. CSR, class switch recombination; DC, dendritic cell; Ig, immunoglobulin;
M cell, Microfold cell; pIgR, polymeric immunoglobulin A receptor; PSA, polysaccharide A; RLR, retinoic acid-
inducible gene-1–like receptor; SFB, segmented filamentous bacteria; SHM, somatic hypermutation; TFH, T fol-
licular helper cells; TH, T-helper cells; TLR, toll-like receptor; Treg, T-regulatory cells.
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Janoff et al
of immunomodulatory FoxP31T-regulatory cells (Treg)
in germ-free mice.17,18Mucosal Treg cells, unlike
thymic Treg cells, have T-cell receptors that are
specific for the antigens of commensal bacteria,
suggesting that local exposure to commensal intestinal
bacterial antigens drives this development.19
Mucosal dendritic cells (DCs) support induction of
immune tolerance by secretion of interleukin-10 that
drives the differentiation of Tregcells. Escherichia coli
or Bacillus subtilis can support differentiation of mono-
cytes into DCs.20DCs can extend through the epithe-
lium to bind microbial glycans in the lumen via
DC-SIGN and other receptors. Intestinal DCs can
present bacterial polysaccharide A of B fragilis to acti-
vate CD41T cells and cytokine secretion.21
Mucosal DCs also initiate the commitment of muco-
sal B cells to immunoglobulin (Ig)A, including gA
specific for the colonizing bacteria.22,23Treg cells,
withT follicular helper
differentiation of mucosal B cells to IgA-producing
plasma cells. Depletion of Tregcells causes reduction
in mucosal IgA production,24as does reversal of bacte-
rial colonization.22These data indicate a functional link
among commensal bacteria, T cells, and induction and
spite the elegant and specific work in mouse models, ef-
forts to establish such direct links between the
microbiome and human immune function in children
and adults are in progress25but are confounded by the
complexity of the human model.
Perturbations ofthedelicate balance betweenimmune
tolerance/ignorance and activation in human disease
states are associated with disturbances in the composi-
tions of commensal communities (ie, ‘‘dysbiosis’’). Al-
tered distributions of various microbial groups have
cancer,31cardiovascular disease,32and human immuno-
deficiency virus transmission.33,34Indeed, microbial
products and metabolites from mucosal sites are found
circulating in the blood35with potential systemic ef-
crobiome influences the human syndrome and the
syndrome influences the microbiome requires robust,
creative, and reproducible study design and analysis.
This issue of Translational Research offers 4 incisive
reviews of our current understanding of the human mi-
crobiome, focusing on the gastrointestinal tract, lung,
female reproductive tract, and virome. Although each
review presents a glimpse into a unique ecological
niche, taken collectively, these articles convey the great
challenges and potentially greater rewards to clinical
practice of elucidating the mechanisms by which the
microbiome affects human health.
plex, yet best studied microbiome, that of the gastroin-
testinal tract. Each section of the digestive tract, from
oral cavity to rectum, provides a different physiochem-
ical environment that is colonized by unique kinds and
models, most notably germ-free mice, has revealed a
plethora of beneficial services that a healthy microbiome
can deliver to its host,37-39including inducing the
development and maintenance of immune homeostasis40
and provision of key nutrients.41As the authors de-
tentially amenable to a range of means of directed
perturbation with the goal of treating or preventing dis-
ease through provision of selected probiotics (Table II),
prebiotics, and antibiotics. Evidence of the clinical effi-
cacy of probiotics and prebiotics is currently lacking in
many instances. However, better understanding of the
functional significance of particular members of a mi-
crobiomecould lead tomorerationalselection ofpoten-
tial probiotic or prebiotic agents. For instance, if a lack
of clostridial species disrupts immune homeostasis and
thus contributestoinflammatory bowel disease,30,42itis
disease. If a canonical set of intestinal microbial
constituents could be determined, successful resolution
of serious chronic recurrent infection with C difficile
might be more amenable to therapy with a microbial
pill, rather than the often successful but cumbersome
fecal transplant from a donor.43
In the second in the series, Beck et al3address a num-
ber of relevant logistic aspects in designing experimen-
tal systems to simulate or inform human biology. They
erate meaningful data on the lung microbiome and its
implications, including standardization between inves-
tigators, the need for appropriate statistical methods,
the pitfalls of small data sets, the need for longitudinal
Table II. Conditions for which probiotics have been
considered as prevention or therapy
Irritable bowel syndrome
Inflammatory bowel disease
Helicobacter pylori–associated gastritis/ulcer disease
HIV disease progression
Liver disease and hepatic encephalopathy
Abbreviations: HIV, human immunodeficiency virus.
Janoff et al
studies to define the stability of the microbiome, and the
potential for confounding by use of different methods.
In addition to the risk of contamination between ana-
tomic sites, they propose that true differences in micro-
bial populations are present within microenvironments
within any tissue, such as the lung, just as geographic
differences will be present between patient groups.
Their consideration of the relevance and challenge of
discriminating between live and dead organisms with
molecular methods is paralleled by an ongoing contro-
versy as to whether noncultivable species identified by
sequencing are relevant as potential pathogens com-
pared with those readily grown in culture. On a patho-
physiologic level, they advance and support the
paradigm that the microbiome may modulate immune
development, immune defense, allergy, inflammation,
and immune tolerance, each of which may be mediated
by specific organisms and mechanisms. Beck et al3do
not see the lung in isolation. Rather, they draw connec-
tions between its microbiome and immunologic respon-
siveness with that of the intestine and propose both
microbiologic and immunologic links between these
otherwise distinct anatomic sites.
With an ecological perspective, Forney et al4focus
less on the specific bacteria but more on their metabolic
products as determinants of the organisms, whether
the lactic acid and associated low pH produced most of-
ten, but not exclusively, by Lactobacillus spp. are high-
lighted as a primary determinant of the microbiota
present or absent in the vagina. An intriguing aspect,
perhaps unique to the vaginal microbiome, is the influ-
ence of hormonal variation from early to adult life,
throughout the menstrual cycle, and with menopause
on the ‘‘vaginal microbial ecosystem.’’ These changes
are reflected in the relative availability of glycogen
locally and thus the presence of organisms capable of
fermenting glycogen to lactic acid. The terms ‘‘rich-
ness,’’ referring to the number of species present, and
‘‘diversity,’’ which also considers the distribution of
these species (Table I) are relevant to their discussion
of how to characterize thevaginal microbiome in differ-
ent conditions. Indeed, geographically and ethnically
distinct women in each population at each anatomic
site and at each time have both shared and distinct mi-
crobial constituents and environmental conditions,
which are also affected by medications, contraceptives,
antibiotics, lubricants, intercourse, and other behaviors.
So, what is the ‘‘normal’’ microbial ecology of the
vagina, how stable and resilient is it over time, and
how can investigators distinguish effects on and from
the host of these microbial populations and their prod-
ucts? In this context the authors highlight that the vagi-
nal microbiota and resultant ecosystem should not be
considered to be in a ‘‘commensal’’ relationship in
which the organisms derive food from but provide no
benefit to the host. Rather, the relationship is one of
‘‘mutualism’’ in which the organisms also provide pro-
tection against colonization and infection of the host by
potentially pathogenic organisms. This perspective is
reinforced by the clinical scenario in which develop-
ment of symptomatic vaginal infections with, for exam-
ple, Candida species follows treatment of urinary tract
infections with antibacterial agents that modify thevag-
In the last article in the series, Wylie et al5tackle the
human virome, the collection of eukaryotic viruses and
bacteriophage (bacterial viruses) that constitute a rela-
tively little studied, yet likely critical, component of
the human-microbe axis. Unlike with bacteria, the lack
of a common genomic element in viruses (eg, a 16S
rRNA gene) greatly complicates novel viral discovery
by necessitating viral enrichment schemes or deep
sequencing. Nevertheless, recent viral metagenomic
surveys performed in a variety of human samples and
disease contexts have revealed a staggering diversity
atively,the identification of myriadhuman and bacterial
genes splicedintoviral andbacteriophagegenomes pre-
dicts a substantial and ongoing flux of genetic informa-
Overall, the reviews in this volume provide a well-
considered overview of the technical hurdles raised by
metagenomic studies, such as which samples to survey,
how best to procure and prepare specimens, how deeply
to interrogate a microbial community, and how to ana-
lyze the data and compare populations. More important,
the authors also confront the theoretic challenge of as-
cribing etiologic significance to the human microbiome
the human microbiome have been noted in many dis-
eases, yet we typically have only circumstantial evi-
dence that the loss or gain of a particular group of
microorganisms actually contributes to disease progres-
microbiome, but to the fact that ‘‘dysbiosis’’ typically is
observed in chronic, multifactorial diseases such as in-
flammatory bowel disease or obesity, and sometimes in
healthy subjects. In these situations, the challenge is to
envision how Koch’s postulates allow one to prove con-
vincingly that a microbiome or metagenome causes dis-
ease orimmuneeffects inhumans,ashasbeen elegantly
and convincingly proven in mice.44-47Rather, the
tenants of risk factor epidemiology can be invoked to
bolster the inferential case in humans. Even the author
of the Bradford-Hill Criteria for assessing evidence of
causation47recognized that the ‘‘criteria,’’ including
Volume -, Number -
Janoff et al
strength of association, consistency in different venues,
specificity of effect, temporality, biological gradient,
and plausibility, were not indisputable evidence for or
against cause and effect. In humans, we must build the
case from several perspectives, such as that advanced
for smoking causing lung cancer or Helicobacter pylori
causing gastric cancer.
plied to the human microbiome, to date, translation of
findings from bench to bedside has proven arduous.
Studies involving human subjects require disentangling
multiple, highly interlinked factors thought to contrib-
ute to disease. We are striving to move beyond ‘‘associ-
ated with’’ and ‘‘may be related to’’ in our efforts to
placethesedata ina meaningful clinical and causalcon-
text. However, the reviews in this volume clearly and
encouragingly indicate that the bedside is informing
the bench. Indeed, the goal of translational research is
to translate, to use and implement results generated
in vitro and in vivo in animals into enhanced under-
standing of clinical human health and disease, and to in-
troduce and test more effective interventions to prevent
or treat disease with behavior, diet, medications, vac-
cines, or modulatory pre- and probiotics (Table II).
The continuing development of metagenomic technol-
ogy for culture-independent interrogation of the human
help us decipher the complex and unique interactions
between ourselves and our microbial world within.
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