C). Similarly to observations on S. Typhimurium,
the pathogen loads in the feces of GF mice, or
of GF mice previously reconstituted with the
microbiota of 4-day-old mice and then orally
infected with C. rodentium, were reduced by 4
to 5 logs after administration of Clostridia (Fig.
3, D and E). No loss of C. rodentium was seen
when a mixture of Bacteroides species was given
to GF mice reconstituted with the microbiota
of neonatal mice (Fig. 3, D and E).
We next asked whether host immunity plays
a role in Clostridia-mediated colonization resistance against S. Typhimurium infection in the
intestine. The microbiota from 4-day-old mice
were transferred to wild-type, mutant GF mice
deficient in Myd88/Trif, two essential adaptors
for signaling via the Toll-like/interleukin-1 (IL-1)/
IL-18 receptor family, or Rag1−/− GF mice that
are devoid of B and T cells. All these reconstituted GF mice exhibited unimpaired colonization resistance against S. Typhimurium infection
upon intragastric administration of Clostridia
compared with GF mice that were not gavaged
with Clostridia (fig. S5). Thus, colonization resistance against S. Typhimurium in the intestine
does not require host stimulation via innate
MyD88/Trif-regulated pathways or adaptive immunity. Certain antimicrobial proteins, including
regenerating islet–derived 3 beta (Reg3b) and IL-
22–induced Reg3g, have been associated with
colonization resistance to pathogens in some systems (13). Notably, the expression of Reg3b, Reg3g,
and Il6, but not Muc2 or Tnfa, was higher in
the cecum of GF mice colonized with the adult
microbiota than in GF mice colonized with the
microbiota of 4-day-old mice (fig. S6). However,
the expression of Reg3b and Reg3g was reduced
in Myd88−/−Ticam−/− GF mice colonized with the
adult microbiota (fig. S6). Likewise, the expression
of Il22, a cytokine involved in the regulation of
intestinal barrier function and Reg3g (14), was
reduced in the intestine of GF mice colonized with
the microbiota of 4-day-old mice compared with
that of adult mice (fig. S6). However, treatment
with a neutralizing antibody to IL-22 to inhibit
IL-22–mediated protection (15) neither affected
S. Typhimurium loads in fecal and cecal contents
nor influenced colon length in infected GF mice
reconstituted with the microbiota of adult mice
To determine whether Clostridia protected
neonatal mice from pathogen challenge, 10-day-old
mice were gavaged with the Clostridia consortium
or left untreated and then intragastrically infected
with the S. Typhimurium DspiA mutant. Notably, ~50% of the neonatal mice inoculated with
S. Typhimurium succumbed to infection, whereas
>90% of the neonatal mice previously gavaged
with Clostridia survived (Fig. 3F). Collectively,
these results indicate that Clostridia mediate colonization resistance against S. Typhimurium and
C. rodentium via a mechanism that is independent of Myd88, Trif, B, and T cells. Furthermore,
administration of Clostridia protects neonatal mice
from mortality induced by pathogen challenge.
With the exception of a few Lachnospiraceae
OTUs, which are present in the microbiota of
12-day-old mice, taxa in the order of Clostridiales
are absent from the microbiota of 4-day-old and
12-day-old mice but robustly colonize the intes-
tine between days 12 and 16 of neonatal life, the
time frame associated with the acquisition of
colonization resistance against pathogens. To as-
sess whether neonatal bacteria promote the colo-
nization of Clostridia species, GF mice were first
colonized with the microbiota from 4-day-old
mice, and 7 days later they were gavaged with
the Clostridia consortium. The abundance of
Clostridium IV and XIVa clusters, which con-
stitute the predominant Clostridia in the con-
sortium assessed by quantitative polymerase
chain reaction (qPCR), was low after intragastric
gavage to GF mice (Fig. 4A and fig. S8A). In the
presence of the 4-day-old neonatal microbiota,
the intestinal colonization of Clostridia increased
by ~6 logs (Fig. 4A and fig. S8A). Thus, coloniza-
tion of Clostridia is reduced in the absence of
neonatal bacteria. However, if GF mice were
reconstituted with the microbiota of 4-day-old
mice, then subsequent intragastric administra-
tion of Clostridia induced robust colonization
resistance against S. Typhimurium (fig. S9). Like-
wise, preinoculation of GF mice with Lactobacillus
murinus or E. coli, species that are present in
4- and 12-day-old neonatal microbiota, respec-
tively, or with Bacteroides acidifaciens whose
colonization coincides with robust acquisition
of Clostridiales in the microbiota of 16-day-old
mice, enhanced the colonization of Clostridia by
5 to 6 logs (Fig. 4B and fig. S8B).
To assess whether bacteria-derived metabo-
lites regulate intestinal colonization by Clostridia,
we performed unbiased capillary electrophoresis–
time-of-flight mass spectrometry (CE-TOFMS)–
based metabolome analysis of the cecal contents
of GF mice and GF mice colonized with dom-
inant bacterial species present in the ceca of
neonatal and adult mice. The metabolome anal-
ysis revealed that amounts of succinate were very
low in the cecal contents of GF mice. Succinate
levels were also low in GF mice reconstituted
with Clostridia, slightly higher in GF mice col-
onized with E. coli, and significantly elevated in
GF colonized with Bacteroides when compared
with GF mice (Fig. 4, C and D). Succinate levels
were increased in GF mice reconstituted with the
microbiota of 12- and 16-day-old mice, but not in
those given microbiota of 4-day-old mice or given
lactobacilli (Fig. 4, D and E), indicating that an
increase in succinate levels is not required for
Clostridia colonization. Administration of succi-
nate, but not acetate or lactate, in drinking water
did, however, enhance colonization of Clostridia
belonging to the dominant IV and XIVa clusters
by 4 to 5 logs (Fig. 4F and fig. S8C). Consistent
with these results, succinate in the drinking water
reduced the intestinal loads of S. Typhimurium
DspiA by ~100-fold in GF mice given the
Clostridia consortium by gavage (Fig. 4G). Aero-
bic and facultative anaerobic bacteria have been
suggested to consume oxygen in the distal intes-
tine, which then promotes the colonization of
strict anaerobes (16). We found that succinate
administration did reduce the concentration of
oxygen in the intestine of GF mice (Fig. 4H).
Together, these results indicate that the neo-
natal microbiota contribute to the acquisition
of protective Clostridia before weaning and is a
critical event that prevents the growth of enteric
pathogens in the gut early in life.
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The authors thank L. Haynes for animal husbandry, D. Peterson for
mouse strains, Genentech for antibody to IL-22, G. Chen and
M. Zeng for critical reading of the manuscript, and the
Germ-Free Animal Core and the Host Microbiome Initiative
at the University of Michigan Medical School for support.
This work was supported by NIH grants DK095782 and DK091191
(G.N.) and AI106302 (C.R.N.); a Career Development
Award from the Crohn’s and Colitis Foundation of America
(Y.-G.K.); fellowships from the Japanese Society for the
Promotion of Science, Kanae Foundation for the Promotion of
Medical Science, and Mishima Kaiun Memorial Foundation
(K.S.); NIH training grant T32DK094775 (J.M.P.); and Grant-in-Aid for
Scientific Research on Innovative Areas “Stem Cell Aging and
Disease” from the Ministry of Education, Culture, Sports, Science and
Technology (15H01522) and the Japan Science and Technology
Agency PRESTO (S.F.). All data and code to understand and assess
the conclusions of this research are available in the main text,
supplementary material, and from the following repositories:
Microbiota data files are available at www.ncbi.nlm.nih.gov/bioproject/
378417 in the National Center for Biotechnology Information (NCBI)
Sequence Read Archive under BioProject PRJNA378417 (SRA:
SRP101509), and the metabolomics data is available from the
Metabolomics Workbench at www.metabolomicsworkbench.org/data/
MS&Result Type=1 (accession number ST000570; Project
PR000418). Clostridia consortium from the University of Chicago
(CL-UC) is available from the University of Chicago under a material
transfer agreement with the University of Chicago. Y.-G.K. and
G.N. conceived and designed experiments. Y.-G.K. and K.S. conducted
most of the experiments, with help from S.-U.S., J.M.P., N.A.P., M.H.,
and X.L. S.F. performed metabolome analysis. T.M.S., E.C.M., T.D. W.,
and C.R.N. provided advice, discussion, and critical materials. T.F.,
A. T.S., and J.M.P. provided critical materials. Y.-G.K., K.S., S.F., N.I.,
and G.N. analyzed the data. Y.-G.K., K.S., and G.N. wrote the
manuscript, with contributions from all authors. C.R.N. is president and
cofounder of ClostraBio, Inc., a company developing microbiome-modulating therapeutics for the treatment of food allergies. Y.-G.K.,
C.R.N., and G.N. are coinventors on patent application 62/442,527,
submitted by the University of Chicago and the University of Michigan,
which is related to the treatment of enteric disease with Clostridia.
Material and Methods
Figs. S1 to S9
23 May 2016; accepted 28 March 2017