Gut biogeography of the bacterial microbiota

Gregory P. Donaldson

S. Melanie Lee

, and

Sarkis K. Mazmanian

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA

91125 USA

PREFACE

Animals assemble and maintain a diverse, yet host-specific gut microbial community. In addition

to characteristic microbial compositions along the longitudinal axis of the intestines, discrete

bacterial communities form in microhabitats, such as the gut lumen, colon mucus layers and colon

crypts. In this Review, we examine how spatial distribution of symbiotic bacteria among physical

niches in the gut impacts the development and maintenance of a resilient microbial ecosystem. We

consider novel hypotheses for how nutrient selection, immune activation and other mechanisms

control the biogeography of bacteria in the gut and discuss the relevance of this spatial

heterogeneity to health and disease.

Humans and other mammals harbor a complex gastrointestinal

microbiota

, which includes

all three domains of life (Archaea, Bacteria and Eukaryota). This extraordinary symbiosis,

formed via a series of exposures to environmental factors, is initiated upon contact with the

vaginal microbiota during birth

. Abrupt changes during the first year of life follow a pattern

that corresponds to gestational age in both mice

and humans

, which suggests that strong

deterministic processes shape the composition of the microbiota during development. These

population shifts may be explained by influences from diet, the developing immune system,

chemical exposures, and potentially founder effects of initial colonizers. Founder effects are

not well understood in the mammalian gut, but the profound changes in host gene expression

that occur in response to microorganisms, and the great potential for

syntrophic

interactions

between bacteria

suggest that early colonizers may have long-term effects on the

establishment of the microbiota. The immune system imposes selective pressure on the

microbiota through both innate and adaptive mechanisms such as antimicrobial peptides

secreted immunoglobulin A (IgA)

, and other contributing factors

(see below). However,

current research suggests that diet may have the greatest impact on microbiota assembly.

Prior to weaning, breast milk plays a crucial part in shaping the microbial community

composition via transmission of the milk microbiota to the infant gut

, protection from

harmful species by secreted maternal antibodies

, and selection for certain species by milk

oligosaccharides, which can be used by microorganisms as carbon sources

. For example, in

in vitro

competitive growth experiments,

Bifidobacterium longum

benefits from its ability to

use fucosylated oligosaccharides that are present in human milk to outgrow other bacteria

that are usually present in the gut microbiota, such as

Escherichia coli

and

Clostridium

Correspondence to: sarkis@caltech.edu.

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perfringens

. Several species of

Bacteroides

can also utilize fucosylated oligosaccharides as

carbon sources

, suggesting that their colonization may be aided by

prebiotic

properties of

milk. Accordingly, children of mothers with nonfunctional fucosyltransferase 2, an enzyme

required for fucosylation of milk oligosaccharides, display lower levels of fecal

Bifidobacteria and

Bacteroides

species

. The importance of diet in determining the

composition of the microbial community in the gut is also highlighted by the observation

that transition to solid foods coincides with establishment of an adult-like microbiota.

The adult intestinal microbiota consists of hundreds to thousands of species, dominated by

the Bacteroidetes and Firmicutes phyla

. This ecosystem is distinct from that of any other

microbial habitats that have been surveyed

, and includes many species that exist nowhere

else in nature, indicating that coevolution of the host with its gut microbial

symbionts

(including

commensals

and

mutualists

) has generated powerful selective mechanisms. A

recent study of how different microbial communities colonize

gnotobiotic

animals showed

that deterministic mechanisms (presumably host-microorganism interactions) led to

reproducible shaping of the microbiota regardless of the source of the input community

The adult intestinal microbiota is also partially stable, as a core of ~40 bacterial species

(accounting for 75% of the gut microbiota in terms of abundance) persists for at least a year

in individuals

. A more extensive longitudinal study found that 60% of all bacterial strains

within an individual persisted for five years

. During severe perturbations such as antibiotic

treatment, the fecal community is depleted to a low-diversity consortium, but after a

recovery period membership and relative abundance largely resemble the pretreatment

state

. Some species that are depleted to undetectable levels in stool are later recovered

suggesting that there may be reservoirs of bacterial cells that can re-seed the intestinal

lumen.

The mucus layer, crypts of the colon and appendix are examples of privileged anatomical

sites, protected from the fecal stream and accessible only to certain microorganisms. In this

Review, we highlight relevant features of spatial heterogeneity of bacterial species and

communities in the gut microbiota, and discuss the impact of microbial localization in

engendering specific and stable colonization with profound implications for health and

disease.

MICROBIAL COMPOSITION OF THE GUT

The mammalian lower gastrointestinal tract contains a variety of distinct microbial habitats

along the small intestine, cecum, and large intestine (colon). Physiological variation along

the lengths of the small intestine and colon include chemical and nutrient gradients, as well

as compartmentalized host immune activity, which are known to influence bacterial

community composition. For example, the small intestine is more acidic, and has higher

levels of oxygen and antimicrobials than the colon (Figure 1A). Therefore, the small

intestine microbial community is dominated by fast-growing facultative anaerobes that

tolerate the combined effects of bile acids and antimicrobials, while still effectively

competing for simple carbohydrates that are available in this region of the gastrointestinal

tract. Bile acids, secreted through the bile duct at the proximal end of the small intestine, are

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bactericidal to certain species due to their surfactant properties and are known to broadly

shape the composition of the microbiota, especially in the small intestine. For example,

feeding mice excess bile acids generally stimulates the growth of Firmicutes and inhibits

Bacteroidetes

. Additionally, the shorter transit time in the small intestine compared to

colon (an order of magnitude shorter, despite the increased length of the small intestine) is

thought to make bacterial adherence to tissue or mucus an important factor for persistent

colonization of the small intestine.

In ileostomy samples from humans, the small intestine was found to exhibit lower bacterial

diversity than the colon, and was highly enriched in certain Proteobacteria and

Clostridium

species

. Furthermore, a metatranscriptomic analysis revealed that the expression of genes

involved in central metabolism and in pathways responsible for import of simple sugars by

facultative anaerobes was greatly enriched in ileal samples, compared to fecal samples

. In

mice, Lactobacillaceae and Proteobacteria (especially Enterobacteriaceae) are enriched in

the small intestine

(Figure 1A). Although bacteria in the small intestine are potentially

competing with the host for nutrients, host-derived bile acids and antimicrobial peptides

limit bacterial growth to low densities in proximal regions. Only at the distal end of the

small intestine (in the terminal ileum) do bacterial densities reach saturating levels similar to

those found in the large intestine (Figure 1A).

The cecum and colon cultivate the most dense and diverse communities of all body habitats.

Mice, like most herbivorous mammals, have a large cecum between the small and large

intestine where plant fibers are slowly digested by the microbiota. Humans have a small

pouch-like cecum with an attached appendix, a thin tube-like extension (Figure 1A). In the

cecum and colon, microorganisms are responsible for the breakdown of otherwise ‘resistant’

polysaccharides that are not metabolized during transit through the small intestine. Lower

concentrations of antimicrobials, slower transit time, and a lack of available simple carbon

sources facilitate the growth of fermentative polysaccharide-degrading anaerobes, notably

those of the high-abundance families Bacteroidaceae and Clostridiaceae. In the mouse, the

cecum is enriched in Ruminococcaceae and Lachnospiraceae, while the colon is enriched in

Bacteroidaceae and Prevotellaceae

. Rikenellaceae are prominent in both the cecum and

colon

. Various host factors drive community differences over the cross-sectional axis of

the gut. The entire wall of the colon folds over itself, creating compartments between folds

(inter-fold regions) that are distinct from the central lumenal compartment (Figure 1B). In

mouse studies that used laser capture microdissection to profile the composition of the

microbial communities in discrete regions, significant differences were observed between

the central lumen compartment and the inter-fold region

. Specifically, the Firmicute

families Lachnospiraceae and Ruminococcaceae were enriched between folds while the

Bacteroidetes families Prevotellaceae, Bacteroidaceae, and Rikenellaceae were enriched in

the digesta

Relative to the digesta, the inter-fold regions are likely to contain greater

amounts of mucus, which can serve as a nutrient source for certain bacteria.

Gut microhabitats: mucus and colon crypts

Throughout the human small intestine and colon, specialized epithelial cells called

goblet

cells

secrete a mucus layer of varying thickness that partially or fully covers the epithelium

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depending on the region, creating a boundary between the gut lumen and host tissue (Figure

2A and 2B). The small intestine harbors a single, tightly-attached mucus layer (Figure 2A),

whereas in the colon, mucus is organized into two distinct layers: an outer, loose layer, and

an inner, denser layer that is firmly attached to the epithelium (Figure 2B). As mentioned

above, bacterial densities are much higher in the colon, compared to the small intestine, and

examination of the colon by fluorescence

in situ

hybridization (FISH) has shown that the

inner mucus layer appears essentially sterile next to the densely populated outer layer

. In

addition to mucus density itself serving as a physical obstacle for microorganisms,

antimicrobial molecules and oxygen secreted from the epithelium accumulate higher local

concentrations within the mucosa, especially in the small intestine, greatly restricting

potential microbial inhabitants.

Mucus is continuously secreted and the outer layers are sloughed off, generating ‘islands’ of

mucus that are carried into the fecal stream

. In mice, a viscosity gradient of the gel-

forming mucus increases from the proximal colon (which includes the cecum and the

ascending and transverse colon) to distal colonic sites (which includes the descending colon

and the sigmoid colon that connects to the rectum). Accordingly, there are more mucus-

associated bacteria in the proximal region

. Mucosal

biofilm

formation in the proximal

colon is conserved from mammals to amphibians

, suggesting an ancient, evolutionarily

conserved origin of this region for interactions with bacteria. Therefore, the mucus layers of

the gastrointestinal tract create environments that are distinct, protected habitats for specific

bacterial ecosystems that thrive in proximity to host tissue.

Divergence between the mucosal and

digesta

associated colonic communities has been

observed in several mammals including humans

, macaques

, mice

, cows

, and flying

squirrels

. More specifically, human colon biopsy and swab samples have revealed a

distinct mucosal community enriched in Actinobacteria and Proteobacteria compared to the

lumen community

. Certain species are highly enriched in colon mucus, such as the mucin-

degraders

Bacteroides acidifaciens

in mice

Bacteroides fragilis

in macaques

and

Akkermansia muciniphila

in mice and humans

(Figure 2B). Human mucosal

communities in biopsy

–

and lavage

samples of the colon contain significant variability

between sample locations less than one centimeter apart, suggestive of the existence of

mucosal microbial populations in patches. Interestingly, an imaging study using approaches

that carefully preserve the structure of feces also identified discrete patches; individual

groups of bacteria were found to spatially vary in abundance from undetectable to saturating

levels

. This spatial niche partitioning in feces may be reflective of aggregates of

interacting microorganisms, heterogeneity of nutrient availability in plant fibers, or

microenvironments in mucosally-associated communities that imprint the digesta as it

transits through the gut. Therefore, microbial profiling of fecal samples, which is the most

common strategy employed in microbiome studies, represents an incomplete and skewed

view of even the colon, which has distinct mucosal communities and spatial heterogeneity

that is lost upon sample homogenization.

Some bacteria completely penetrate the mucus and are able to associate directly with the

epithelium, within the crypts of the colon. Crypt-associated microorganisms were first

described using electron microscopy

. Many subsequent imaging studies likely failed to

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observe or underestimated the number of tissue-associated bacteria because common

washing and fixing methods can remove mucosal biofilms

. This led to the hypothesis that

the mucosal surface is largely devoid of microbial colonization in healthy individuals.

However, imaging studies using Carnoy’s fixative, which is known to preserve the mucosal

layer, found that there are bacteria in a significant fraction of colonic crypts in healthy

mice

and humans

. More recent work using laser microdissection and sequencing to

profile mouse crypt-associated communities revealed that the community is especially

dominated by

Acinetobacter

spp. and is generally enriched for Proteobacteria capable of

aerobic metabolism

(Figure 2B). Evasion of immune responses and particular metabolic

activities are likely required for crypt occupancy by microorganisms specialized to reside in

close proximity to the host. A well-characterized example of this adaptation is the ability of

the human symbiont

B. fragilis

to enter crypts of the proximal colon of mice via a process

requiring both modulation of the immune system

and utilization of specific host-derived

nutrients

(see below). While dogma has emerged that microorganisms contact mucosal

surfaces exclusively in disease states, it appears that life-long physical associations between

specific members of the microbiota and their hosts represent symbioses forged over

millennia of co-evolution.

MECHANISMS RESPONSIBLE FOR GUT BIOGEOGRAPHY

Several factors influence the biogeography of bacteria within the gut, including diet,

antimicrobials, mucus and adherence, and the host immune system.

Diet and nutrients

Bacterial metabolism in the gut likely contributes to the localization of particular groups of

microorganisms. Because fatty acids and simple carbohydrates from food are absorbed and

depleted during transit through the small intestine, sustainability of the colonic bacterial

ecosystem requires growth by fermentation of complex polysaccharides, the principal

carbon sources that reach the colon. Best studied in this regard are

Bacteroides

species,

which are able to catabolize polysaccharides derived from the diet and from the host

Compared to other gut bacteria,

Bacteroides

have the largest number and diversity of genes

involved in polysaccharide degradation

. This extensive array of polysaccharide utilization

systems is dominated by those resembling the starch utilization system (Sus), originally

described in

Bacteroides thetaiotaomicron

. Sus systems consist of lipid-anchored

enzymes either secreted or displayed on the bacterial cell surface that can catabolize

particular complex glycans into smaller oligosaccharides, which are then imported through a

dedicated outer membrane transporter (Figure 3A). In the gut,

Bacteroides

species use Sus-

like systems to break down dietary polysaccharides and host-derived mucin glycans

. The

genome of

B. thetaiotaomicron

encodes 88 Sus-like systems presumably with different

glycan specificities, providing remarkable metabolic flexibility

. Based on these findings,

Bacteroides

species, and

B. thetaiotaomicron

in particular, are sometimes referred to as

“generalists,” capable of occupying a variety of metabolic niches depending on the

availability of diverse polysaccharide nutrients.

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Diet-derived polysaccharides control microbial community composition in the lumen of the

colon. Unsurprisingly, the influence of diet is readily apparent in studies that profile the fecal

community. A study of humans that completely switched between plant and animal-based

diets showed that the microbiome abruptly shifts with diet

. Over small time scales this

effect is reversible, suggesting that these changes represent transient ecosystem adaptations

via blooms of particular species in the lumen while the mucosal reservoir remains

unchanged. Many studies of

Bacteroides

glycan metabolism have shown that restricting the

polysaccharide content of the mouse diet allows selection for species (or strains) that are

capable of metabolizing the complex glycans present, such as fructans

, human milk

oligosaccharides

, fucosylated mucin glycans

, and mannan

. Presumably, the variety of

Sus-like systems present in the genomes of

Bacteroides

provides the metabolic plasticity to

persist in the gut despite short and long-term changes in nutrient availability. However, even

in terms of monosaccharide and disaccharide utilization, there is a hierarchy of bacteria that

are more efficient consumers, which helps explain how diet can dramatically and rapidly

change the composition of the fecal community. Importantly, the nutrient environment of the

gut lumen may be in a dynamic state of flux due to potential meal-to-meal variability,

especially in omnivorous mammals.

In contrast to the variable conditions in the gut lumen, mammals likely maintain a more

consistent nutrient balance in the mucosa, which serves as a stable positive selection factor

for certain species of bacteria. Mucus degradation and metabolism by gut microorganisms

provides access to privileged spatial niches and therefore a competitive advantage over other

species, both

indigenous

and invasive. For example, several studies have shown that the

ability to grow in an

in vitro

mucus culture is generally predictive of the ability of a bacterial

species to colonize the mouse gut

MUC2

alone is coated with over 100 different O-

linked glycan structures in humans

. These glycans differ between mice and humans

, and

differences in complex glycan “preference” by various bacterial species is a suggested

mechanism of host-specific selection of a characteristic microbiome profile. In agreement,

computational models have shown that positive selection at the epithelium via the ability to

metabolize specific nutrients can be a more powerful mechanism for shaping host-associated

microbial communities than negative selection driven by antimicrobials

A. muciniphila

, a prominent symbiont in many mammals, is one of the most effective mucin

degraders

in vitro

and is consistently found at high abundance in the mucus layer in

humans

and mice

. Consumption of mucus glycans as a carbon and energy source allows

A. muciniphila

and other mucin-degraders to colonize the gut independently of the animal’s

diet, providing a clear advantage to the bacteria during conditions of nutrient deprivation.

Accordingly, levels of

A. muciniphila

increase in fasting Syrian hamsters

and hibernating

ground squirrels

. Similarly, during intestinal inflammation in mice, the community

metatranscriptome indicates increased mucin utilization with a corresponding increase in

abundance of the mucin-degrading

B. acidifaciens

. In gnotobiotic mice, restriction of

complex polysaccharides in the diet causes the generalist

B. thetaiotaomicron

to shift its

metabolism to utilize mucin glycans

. Further work has revealed that mutations in Sus-like

systems involved in mucin glycan utilization in

B. thetaiotaomicron

cause a defect in

competitive colonization and in vertical transmission of bacteria from mother to pup

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Therefore, the ability to utilize mucus as a carbon and energy source contributes to the

ability of some microorganisms to stably colonize the host and transfer to offspring across

generations. Not surprisingly, genetic manipulation of enteric mucus production in mice

changes microbial community composition

. In turn, gut bacteria affect transcription of

mucin-encoding genes in mice

. Overall, development of a healthy mucosa is a

collaborative, bi-directional event between the host and the gut microbiota, creating an

environment that allows the specific members to establish persistent colonization via

utilization of host-derived glycans.

In some cases, the ability of a bacterium to colonize the gut may be determined by its ability

to utilize a specific, yet limiting, nutrient. Bacterial species-specific carbohydrate utilization

systems termed commensal colonization factors (CCFs) have been identified in

B. fragilis

and

Bacteroides vulgatus

, and allow these bacteria to colonize saturable nutrient niches

This discovery was made based on the observation that gnotobiotic mice colonized with a

specific

Bacteroides

species are resistant to colonization by the same species, but not

colonization by closely related species. A genetic screen revealed that a set of genes

encoding the CCF system was required for this intra-species

colonization resistance

phenotype (Box 1), suggesting that CCFs are responsible for defining the species-specific

niche. Accordingly, when the

ccf

genes from

B. fragilis

were expressed in

B. vulgatus

, the

resulting hybrid strain gained the ability to colonize an alternate niche. The CCF system was

also required for penetration of

B. fragilis

into the crypts of the colon and long-term

resilience to intestinal perturbations such as antibiotic treatment and gastroenteritis.

Collectively, these data suggest that while metabolic flexibility allows bacterial adaptation in

the lumen environment, the occupation of a narrowly-defined, tissue-associated niche is

likely very important for stable colonization by some bacteria.

Box 1

Colonization resistance

One of the benefits afforded by the microbiota to the host is colonization resistance to

pathogens. Invasive species of bacteria are inhibited from colonizing the gut because they

are unable to displace indigenous species that have gained a strong foothold. After years

of studying colonization resistance against pathogens in gnotobiotic animals in the

1960’s and 70’s, Rolf Freter theorized that the ability of a bacterial species to colonize

the gut is determined by its ability to utilize a specific, limiting nutrient

135

. This notion

has been well supported by studies showing that colonization resistance to pathogens is

mediated by the availability of nutrient niches in the cases of

Escherichia coli

136

and

Clostridium difficile

137

. But Freter’s hypothesis reached even further, suggesting that the

relative amounts of limiting nutrients could dictate the abundance of each species in the

indigenous community. Correspondingly, the variety of host-derived growth substrates

could explain the stable diversity of the gut microbiota if individual species have evolved

to specialize in the uptake and metabolism of specific, limiting nutrients, such as in the

case of

Bacteroides fragilis

. The concept of spatial niche partitioning being governed

by host production of specific and scarce nutrient resources is attractive, and may help

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explain both long-term persistence and resilience of the microbiome, as well as

colonization resistance to pathogens.

Antimicrobials

Specialized epithelial immune cells called

Paneth cells

reside at the base of the crypts of the

small intestine, secreting an array of antimicrobials that restrict the growth of bacteria that

are found near the mucosal surface

. Many of these molecules are cationic antimicrobial

peptides that interact with and disrupt negatively charged bacterial membranes (Figure 3B).

Modifications to lipid A, a major component of the outer membrane of gram-negative

bacteria, are known to confer resistance to cationic antimicrobial peptides in several

pathogens

. Interestingly, underphosphorylation of this lipid portion of LPS, a modification

shared with the

pathobiont

Helicobacter pylori

, was found to be important for resilient

colonization by

B. thetaiotaomicron

during inflammation

(Figure 3B).

The concentration of a variety of antimicrobials is higher toward the proximal end of the

small intestine, creating a gradient that leads to a higher abundance and diversity of bacteria

in distal locations (Figure 1A). For example, the lectin RegIII

is bactericidal to gram-

positive bacteria that dominate the small intestine because it binds to and disrupts their

exposed peptidoglycan layer. RegIII

is required to prevent massive infiltration of the

mucosa and microbial invasion of the tissue

. In addition to RegIII

, the innate immune

system deploys many other antimicrobials (

such as alpha-defensins from Paneth cells and

beta-defensins from neutrophils)

with differing specificities to limit access to the

epithelium

, and resistance to these host-derived antimicrobial peptides is a general feature

of many indigenous gut species of Firmicutes and Bacteroidetes

In addition to these antimicrobials, gut bacteria, which are largely anaerobic, must contend

with reactive oxygen species produced by aerobic host metabolism. Rapid dilution and

consumption of oxygen secreted from the host tissue generates a gradient of oxygen that

decreases in concentration from tissue to lumen (Figure 2). Accordingly, the mucosal

community is enriched in genes required for resistance to reactive oxygen species

Notably, although all

Bacteroides

species are classified as obligate anaerobes,

B. fragilis

can

use oxygen as a terminal electron acceptor at nanomolar concentrations

B. fragilis

and

tissue-associated

microaerophilic

Lactobacillaceae express catalase, superoxide dismutase,

and other enzymes to inactivate reactive oxygen species

. Altogether, these mechanisms

restrict access to the epithelium to a subset of bacterial species that not only can utilize

nutrients found only at the tissue boundary, but can survive host antimicrobial strategies as

well.

Mucus and adhesion

To access the epithelium, pathogens and commensals alike must contend with the mucus

barrier and the immune system (Figure 4). Secreted MUC2 forms peptide crosslinks to

create a viscous gel-like substance

, serving as a barrier and host defense mechanism

. In

mice lacking MUC2, the crypts of the colon are filled with bacteria and the tissue is covered

in biofilms

, indicating that the gel-forming mucus is the primary barrier to tissue

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