Molecular Psychiatry (2016), 1–11

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ORIGINAL ARTICLE
Gut microbiome remodeling induces depressive-like behaviors
through a pathway mediated by the host’s metabolism
P Zheng1,2,3,8, B Zeng4,8, C Zhou1,2,3,8, M Liu1,2,3, Z Fang1,2,3, X Xu1,2,3, L Zeng1,2,3, J Chen1,2,3, S Fan1,2,3,
X Du1,2,3, X Zhang1,2,3, D Yang5, Y Yang1,2,3, H Meng6, W Li4, ND Melgiri1,2,3, J Licinio7,9, H Wei4,9 and P Xie1,2,3,9

Major depressive disorder (MDD) is the result of complex gene–environment interactions. According to the World Health
Organization, MDD is the leading cause of disability worldwide, and it is a major contributor to the overall global burden of disease.
However, the definitive environmental mechanisms underlying the pathophysiology of MDD remain elusive. The gut microbiome is
an increasingly recognized environmental factor that can shape the brain through the microbiota-gut-brain axis. We show here that
the absence of gut microbiota in germ-free (GF) mice resulted in decreased immobility time in the forced swimming test relative to
conventionally raised healthy control mice. Moreover, from clinical sampling, the gut microbiotic compositions of MDD patients
and healthy controls were significantly different with MDD patients characterized by significant changes in the relative abundance
of Firmicutes, Actinobacteria and Bacteroidetes. Fecal microbiota transplantation of GF mice with ‘depression microbiota’ derived
from MDD patients resulted in depression-like behaviors compared with colonization with ‘healthy microbiota’ derived from
healthy control individuals. Mice harboring ‘depression microbiota’ primarily exhibited disturbances of microbial genes and host
metabolites involved in carbohydrate and amino acid metabolism. This study demonstrates that dysbiosis of the gut microbiome
may have a causal role in the development of depressive-like behaviors, in a pathway that is mediated through the host’s
metabolism.

Molecular Psychiatry advance online publication, 12 April 2016; doi:10.1038/mp.2016.44

INTRODUCTION further investigation in larger, well-characterized MDD popula-

Major depressive disorder (MDD) is a debilitating mental disorder tions is still required, as these previous studies have not addressed
affecting up to 15% of general population and accounting for whether disturbances in gut microbiota have a causative role in
12.3% of the global burden of disease.1 MDD increases health- the onset of MDD.
care expenditures and suicide rates.2 In recent decades, several In recent years, mounting evidence has demonstrated that gut
theories have attempted to explain the pathogenesis of MDD, microbiota can greatly influence all aspects of physiology.14
including neurotransmission deficiency,3 neurotrophic alterations,4 Variations in the composition of gut microbiota have been
endocrine-immune system dysfunction5 and neuroanatomical reported to have crucial roles in the pathogenesis of several
abnormalities.6 As none of these theories has been universally enteric and metabolic diseases, such as irritable bowel syndrome,
accepted, a definitive pathogenesis of MDD remains largely diabetes and obesity.15–19 Emerging evidence also suggests that
elusive, and there is a pressing need to identify novel pathophy- gut microbiota can influence brain function and behavior through
siologic mechanisms underlying the disorder. the ‘microbiota-gut-brain axis’.20–24 Germ-free (GF) mice, which
Previously, we found that MDD was associated with obvious are devoid of any bacterial contamination, have been widely used
disturbances in peripheral and central metabolites.7–10 Interest- to investigate such phenomena.24 Recent studies have demon-
ingly, several altered metabolites in MDD subjects (such as strated that GF mice display reduced non-spatial memory, social
hippurate, dimethylamine and dimethylglycine) are metabolic motivation and anxiety compared with their conventionally raised
byproducts of gut microbiota.9 Moreover, previous clinical studies specific pathogen-free (SPF) counterparts.21,23,25,26 Another recent
have reported disturbances in the gut microbiotic compositions in study has described that targeted modifications in gut microbiota
limited samples of heterogeneous depressed subjects with similar can correct autism spectrum disorder-related behavioral
findings observed in animal models of depression.11–13 These abnormalities.27 On the basis of these findings, we hypothesized
preliminary studies highlight the potential association of gut that the dysbiosis of gut microbiota may be a contributory factor
microbiotic changes and the development of MDD. However, to the development of depression.

1
Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 2Chongqing Key Laboratory of Neurobiology, Chongqing, China;
3
Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing, China; 4Department of Laboratory Animal
Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China; 5Department of Neurology, Yongchuan Hospital, Chongqing Medical University,
Chongqing, China; 6Department of Psychiatry, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China and 7Mind & Brain Theme, South Australian Health
and Medical Research Institute and Department of Psychiatry, School of Medicine, Flinders University, Adelaide, SA, Australia. Correspondence: Professor H Wei, Department of
Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Gaotanyan Street, Chongqing 400038, China or Professor P Xie, Department of
Neurology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuzhong District, Chongqing 400016, China or Professor Julio Licinio, Mind & Brain Theme,
South Australian Health and Medical Research Institute and Department of Psychiatry, School of Medicine, Flinders University, Adelaide, SA, Australia.
E-mail: [email protected] or [email protected] or [email protected]
8
These authors contributed equally to this work.
9
These authors are co-senior authors.
Received 8 June 2015; revised 15 February 2016; accepted 17 February 2016
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In this study, we initially assessed how gut microbiota history of systemic medical illness or mental disorders or family history of
physiologically influence the psychobehavioral characteristics of any psychiatric disorder. A total of 58 MDD patients and 63 demographi-
GF and SPF mice. Then, using 16S rRNA gene sequencing, the gut cally matched healthy controls were recruited from the psychiatric center
microbial communities of 58 MDD patients and 63 healthy and medical examination center of the First Affiliated Hospital at
Chongqing Medical University, respectively. The majority of MDD subjects
controls were compared to evaluate whether alterations in the gut
(n = 39) were drug-naive, while the remaining MDD subjects (n = 19) were
microbiome are associated with MDD status. Furthermore, to being treated with various anti-depressants. All MDD subjects and healthy
assess whether alterations of gut microbiota have a causal role in controls who were using antibiotics or prebiotics were excluded. The
depression-like behavior, gut microbiome remodeling was accom- detailed characteristics of these recruited subjects are shown in
plished through fecal microbiota transplantation (FMT) from either Supplementary Table S1.
MDD patients or healthy individuals on GF mice followed by
behavioral testing to assess depression-like behaviors. Finally, Fecal sample collection and 16S rRNA gene sequencing
metagenomic and metabolomic analyses of samples from the Fecal samples were collected from the recruited subjects or FMT model,
mice harboring ‘depression microbiota’ were conducted to frozen immediately following collection and stored at − 80 °C prior to
examine how the gut microbiome influences host metabolism. analyses. Fecal samples were pulverized with a mortar and pestle in liquid
nitrogen, and bacterial genomic DNA was extracted by the standard Power
Soil Kit protocol. Briefly, the fecal samples were thawed on ice. The MoBio
MATERIALS AND METHODS lysis buffer was added to these fecal samples, which were further vortex
Behavioral testing mixed. Fecal suspensions were centrifuged and the supernatant placed
The protocols of animal experimentation were reviewed and approved by into the MoBio Garnet bead tubes containing MoBio buffer.
the Ethical Committee of Chongqing Medical University (ECCMU, Roche 454 sequencing (454 Life Sciences Roche, Branford, PA, USA): The
Chongqing, China) and the Third Military Medical University (Chongqing, V3-V5 regions of the 16S rRNA gene extracted from the fecal samples of
recruited subjects and cecum samples from recipient mice at 2 weeks post
China). Male GF Kunming mice and SPF Kunming mice were bred in the
FMT were PCR-amplified with barcoded universal primers containing linker
Experimental Animal Research Center at the Third Military Medical
sequences for 454-pyrose-quencing.35
University. GF mice were kept in flexible film gnotobiotic isolators until
Illumina MiSeq sequencing (San Diego, CA, USA): The V4-V5 regions of
the beginning of experiments. All animals were fed the same autoclaved
the 16S rRNA gene extracted cecum samples from recipient mice at 1 week
chow and water ad libitum under a 12-h light-dark cycle (lights on
post FMT were PCR-amplified with primers containing linker sequences for
at 0730) and a constant temperature of 21–22 °C and humidity of 55 ± 5%.
Illumina MiSeq sequencing.36
For each test, the mice were transferred to the experimental room for
acclimation at least 1 h prior to behavioral testing. All tests described
below were carried out by observers blind to the animal genotypes 16S rRNA gene sequencing analysis
between 0800 and 1700. All behavioral tests were videotaped and Raw sequences obtained from 454 sequencing were quality-filtered using
quantified by a video-computerized tracking system (SMART, Panlab, Mothur (Version 1.31.2, http://www.mothur.org/) to obtain unique reads.37
Barcelona, Spain).28 Sequences of less than 200 bp and greater than 1000 bp as well as
Open-field test (OFT): All mice were individually tested in an open-field sequences containing any primer mismatches, barcode mismatches,
apparatus29 consisting of a black square base (45 × 45 cm2) with black ambiguous bases and homopolymer runs exceeding six bases were
walls (45 cm in height). A single mouse was gently placed in the corner of excluded. Raw sequences obtained from MiSeq sequencing were quality-
the chamber, and after 1 min of adaptation, all spontaneous activities were filtered using QIIME (version 1.17) with the following criteria: (i) 300-bp
recorded for 5 min using the video-computerized tracking system. The reads were truncated at any site receiving an average quality score of less
total motion distance was used as an index of locomotor activity, while the than 20 over a 50-bp sliding window and discarding the truncated reads
proportion of distance spent in the center (inner 25% of the surface area) that were shorter than 50 bp; (ii) exact barcode matching, two-nucleotide
was construed as an index of anxiety-like behavior. mismatch in primer matching and reads containing ambiguous characters
Y-maze: The Y-maze apparatus30 consisted of three dark gray arms (45 cm were removed; and (iii) only sequences that overlapped longer than 10 bp
in length × 10 cm in width × 29 cm in height). Each mouse was placed at the were assembled according to their overlap sequence.
end of one arm and allowed to freely explore the maze for 8 min. The All remaining sequences were assigned to operational taxonomic units
sequence and total number of arms entered was recorded. Entry into an arm (OTUs) with a 97% threshold of pairwise identity and then classified
was considered valid only when all four paws of the mouse were inside that taxonomically using the RDP reference database (http://www.mothur.org/
arm. The percentage of alternation was the number of triads containing wiki/RDP_reference_files).38 These taxonomies were used to construct
entries into all three arms divided by the maximum possible number of summaries of the taxonomic distributions of OTUs, which can then be
alternations (the total number of arms entered minus 2) × 100%. applied to calculate the relative abundances of microbiota at different
Tail suspension test (TST): Mice were individually suspended by their levels. Alpha diversity was calculated by four different parameters: (i)
tails31 using adhesive tape (distance from tip of tail was 2 cm). Test observed species; (ii) Shannon Index; (iii) phylogenetic diversity and (iv)
sessions lasted for 6 min with the last 5 min scored for immobility. Mice Simpson.39,40 Distance matrices (Beta diversity) between samples were
that climbed on their tails were removed from further testing. Animals generated on the basis of weighted (Bray-Curtis similarity) and non-
were considered to be immobile when they exhibited no body movement weighted (unweighted UniFrac) algorithms and reported according to
and hung passively. principal coordinate analysis (PCoA).41 To perform the UniFrac analysis,
Forced swimming test (FST): The mice were placed individually in a representative sequences for each OTU were aligned using PyNAST, and a
Plexiglas cylinder (30 cm in height × 15 cm in diameter) filled with 15 cm phylogenetic tree from this alignment was constructed with Fast Tree.42
water (24 ± 1 °C).32 Immobility was defined as the absence of all motion Random Forest algorithm was carried out to identify the key discriminatory
with the exception of movements required to keep the mouse’s head OTUs,43 which assigns an importance score to each OTU by estimating the
above water. Test sessions lasted for 6 min with the last 5 min scored for increase in error caused by removing that OTU from the set of predictors.
immobility.
FMT
Subject recruitment and sample collection Fecal samples from randomly chosen MDD patients (n = 5, male, age 27–61
The protocols of clinical experimentation were reviewed and approved by years) and healthy controls (n = 5, male, age 29–62 years) were used to
ECCMU. Written informed consent was obtained from all recruited human colonize the guts of GF mice. The procedures of preparing the fecal
subjects. Recruitment of MDD and healthy subjects was performed as samples for microbiota transplantation were as described in a previous
previously described.7,9 Briefly, MDD diagnoses were carried out according study.16 Briefly, fecal samples were handled under anaerobic conditions.
to the Structured Psychiatric Interview using DSM-IV-TR criteria,33 and the Each fecal sample (0.1 g) was suspended with 1.5 ml of reduced sterile
17-item Hamilton Depression Rating Scale was used to quantify the phosphate-buffered saline, and pools were made from equal volumes of
severity of MDD.34 MDD candidates were excluded on the basis of donor suspensions. Adult (6–8-week-old) male GF Kunming mice were
substance abuse in addition to pregnancy, nursing or current menstruation colonized with pooled samples derived from either MDD patients or
for female subjects. Healthy controls were excluded on the basis of a healthy controls. The ‘depression microbiota’ and the ‘healthy microbiota’

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recipient mice were separately bred in different gnotobiotic isolators to v2.2.29+) search against the KEGG GENES Database v58 and eggNOG
prevent normalization of gut microbiota. Within each individual gnotobiotic v3.0 database with an E-value cutoff of 1e-5. Bitscore (greater than or equal
isolator, either ‘depression microbiota’ or ‘healthy microbiota’ recipient to 60) and minimum alignment length (greater than or equal to 15 aa)
mice were bred in different cages (five mice per cage). The weights of the were then used to filter the blast hits.54 Linear discriminant analysis was
mice were measured at the beginning of FMT experimentation and used to identify the differential KEGG pathway and KEGG enzyme
immediately prior to killing of the mice. The behavioral tests (including OFT, commission number (E.C.s) representation between fecal microbiomes of
FST and TST) were performed on weeks 1 and 2 after microbiota the humanized depressed and healthy control mice.55
transplantation. Cecal samples were collected at the time the mice were
killed and immediately snap-frozen in liquid N2 and stored at − 80 °C.
RESULTS
Comparisons of metabolite profiles from the FMT model Absence of gut microbiota produces decreased immobility time in
On week 2 post FMT, the depressed and control mice were killed, and the FST
cecum, serum and hippocampus samples were obtained. These samples We initially assessed how the absence of gut microbiota
were subsequently extracted and analyzed by gas chromatography-mass physiologically influences psychobehavioral characteristics by
spectrometry (Agilent 7890A/5975C (Agilent Technologies, Santa Clara, CA, comparing GF mice with SPF mice. From the OFT, representative
USA)), liquid chromatography-mass spectrometry (Agilent 6538 UHD and motion tracks for a GF mouse and a SPF mouse are presented in
Accurate-Mass Q-TOF/MS) and nuclear magnetic resonance (Bruker
AVANCE II 600, Bruker Biospin, Rheinstetten, Germany), based metabolo-
Figures 1a and b. We found no difference in total motion distance
mics platforms. between GF and SPF mice (Figure 1c). In contrast, the proportion
Gas chromatography-mass spectrometry metabolite profiles were of central motion distance was significantly increased in GF mice
processed according to our previously published work.44 The resulting relative to SPF mice (Figure 1d), suggesting reduced anxiety-like
three-dimensional matrix—including peak indices (RT-m/z pairs), sample behavior in GF mice. In the Y-maze spontaneous alternation test,
names (observations) and normalized peak area percentages—were the alternation percentage was significantly decreased in GF mice
introduced into SIMCA-P 12.0 (Umetrics, Umeå, Sweden). Multivariate relative to SPF mice, indicating that GF mice displayed better
statistical methods, such as partial least squares discriminant analysis (PLS- memory performance compared with SPF mice (Figure 1e). In the
DA), were used to identify differential metabolites between groups.45 The
FST, immobility time is widely used as an index of depression-like
differential metabolites were identified using a statistically significant
threshold of variable influence on projection values obtained from the PLS- behavior. Here, we found that GF mice displayed decreased
DA model and a two-tailed Student’s t-test. Metabolites with variable depression-like behavior as evidenced by a significantly decreased
influence on projection values of greater than 1.0 and P-values of less than immobility time (Figure 1f). Expanding on previous studies that
0.05 were deemed statistically significant.46 The Human Metabolome have observed altered anxiety and memory states from changes in
Database was used to comprehensively analyze the differential metabo- gut microbiotic composition,56–58 we have for, we believe, the
lites in terms of in vivo metabolic activity. first time found that the absence of gut microbiota decreases
Nuclear magnetic resonance-based metabolomics analysis of cecum immobility time in the FST, suggesting a potential link between
samples: a 70-mg fecal sample was extracted with 700 μl of phosphate the ‘microbiota-gut-brain axis’ and depression-like behavior.
buffer solution (phosphate-buffered saline, 0.1 M, pH 7.4). Then, 400 μl
samples of supernatant were mixed with 200 μl of phosphate-buffered
saline. After centrifugation at 12 000 r.p.m. for 10 min, 500 μl samples of MDD patients exhibit significant alterations in their gut
supernatant were transferred into 5-mm nuclear magnetic resonance microbiomes
tubes. The proton spectra were collected on a 600 Spectrometer operating Given clinical metabonomic observations of metabolic distur-
at 599.925 MHz 1H frequency. A standard NOESYPR1D pulse sequence was
bances generated from changes in gut microbiota in addition to
used (recycle delay-90o-t1-90o-tm-90o-acquire free induction decay). PLS-
DA was used to visualize discrimination between healthy controls and
murine studies showing that the absence of gut microbiota
MDD subjects.45 The coefficient loading plots of the model were used to influences depression-like behavior, we next sought to determine
identify the spectral variables responsible for sample differentiation on the whether MDD patients displayed disturbances in their gut
scores plot. A correlation coefficient (|r|) of greater than 0.500 was used as microbiomes. The stool samples, as well as demographic and
the cutoff value for statistical significance based on a P-value of 0.05. clinical data, from 58 MDD patients and 63 demographically
Liquid chromatography-mass spectrometry-based metabolomics analy- matched healthy controls were collected. The detailed character-
sis of cecum samples: an 80-μl aliquot of serum sample was mixed with istics of these recruited subjects are shown in Supplementary
240 μl of methanol. Then, 0.01 g of hippocampal tissue was extracted with Table S1. There were no significant differences in their key
1000 μl of buffer (chloroform/methanol = 2:1). The extracted supernatants
demographic characteristics (Supplementary Table S2).
of serum and hippocampus were subjected to Agilent 6538 UHD and
Accurate-Mass Q-TOF/MS. Spectra were collected in both positive and A culture-independent, 16S ribosomal RNA gene-sequence-
negative ESI mode. PLS-DA were used to identify differential metabolites in based approach was used to compare the gut microbial commu-
GF mice relative to SPF mice.45 The differential metabolites were identified nities of MDD patients and healthy controls. DNA was extracted
with variable influence on projection values of greater than 1.0 and from their fecal samples. V3-V5 variable region of bacterial 16S rRNA
P-values of less than 0.05. genes was PCR-amplified. Samples were multiplexed and pyrose-
quenced, followed by quality filtering and chimera checking. We
Shotgun metagenomic analysis of cecum samples obtained a total of 854 639 high-quality 16S rRNA gene sequences
Samples were sequenced by Illumina HiSeq2500. Raw datasets of PE read (7063 ± 2352 reads/fecal sample, Supplementary Table S3), which
files were run through Trimmomatic (v0.32) to remove low-quality base were subsequently clustered into OTUs at a 97% similarity level. The
pairs and sequence adapters using these parameters [SLIDINGWIN- majority of these OTUs belonged to only two phyla (Firmicutes and
DOW:4:15 MINLEN:36].47 Trimmed reads were filtered using the Fastq Bacteroidetes; 83.1 ± 11.9%).
quality filter program (Fastx toolkit v0.0.13.2 ) with the parameters [-q 10 -p Initially, the within-sample (α) phylogenetic diversity analysis
10].48 MetaPhlAn v1.7.8 and Bowtie2 v2.2.1 were used for profiling the showed that there were no significant difference between the two
taxonomic clades in the high-quality metagenomic datasets.49,50 The groups (Supplementary Figures S1a–d; Supplementary Table S4).
paired-end and singleton reads were assembled using an IDBA-UD v1.1.1 In addition, the unweighted UniFrac analysis—which focuses on
assembler.51 The open reading frames of the assembled scaffold sequence
were annotated using Prodigal v2.60 gene finder with the parameter [-p
the degree of microbial phylogenetic similarity (β-diversity)—was
meta].52 Bowtie2 v2.2.1 aligner was used to map the reads to the used to determine the degree by which the gut microbiota
assembled scaffold. The method for estimating the abundance of each within MDD subjects differed from those within healthy controls.
predicted open reading frame from a sample has been previously The three-dimensional plots of unweighted UniFrac analysis
described.53 KEGG Orthology was assigned through BLAST (BLASTP showed an obvious difference in the gut microbial community

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Figure 1. Effect of gut microbiota on mood-related behavior. (a, b) Representative motion tracks for a germ-free (GF) mouse and a specific
pathogen-free (SPF) mouse. (c) Open-field test (OFT): the total motion distance was measured to assess locomotor activity. There was no
difference in total motion distance between GF and SPF mice (n = 22/GF group; n = 15/SPF group). (d) OFT: the proportion of central motion
distance was quantified to assess anxiety-like behavior. GF mice displayed an increased proportion of central motion distance relative to SPF
mice (n = 22/GF group; n = 15/SPF group). (e) Y-maze test: alternation rate of GF mice was significantly decreased compared with SPF mice
(n = 17/GF group; n = 20/SPF group). (f) Forced swimming test (FST): GF mice displayed decreased depression-like behavior as evidenced by a
significant decreased immobility time (n = 21/GF group; n = 15/SPF group). Data presented as means ± standard errors of the mean. **Po 0.01,
***P o0.001 by t-test.

compositions between MDD patients and healthy controls To identify the gut microbiota primarily responsible for
(Figure 2a). A similar discrimination between MDD and healthy discriminating MDD subjects from healthy controls, we applied a
control group was also observed using weighted UniFrac Random Forests classifier, which assigns an importance score to
analysis (Supplementary Figure S2). These differences in the each OTU by estimating the increase in error caused by removing
gut microbiomes were not significantly related to any key that OTU from the set of predictors. A total of 54 OTUs whose
categorical variables (that is, sex, smoking status and antidepres- relative abundance reliably distinguished MDD and healthy
sant use, Supplementary Figures S3a–e) nor to any key continuous control samples were identified (Figure 2b, Supplementary Table
variables (that is, age and body mass index, Supplementary S5). Of these 54 differential OTUs, a total of 29 OTUs were
Figures S4a and b). overrepresented in MDD subjects and assigned to the families of

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Figure 2. 16S rRNA gene sequencing reveals changes to microbial diversity in MDD. (a) Three-dimensional principal coordinate analysis (PCoA)
of unweighted UniFrac distances showed an obvious difference in gut microbiotic composition between major depressive disorder (MDD)
patients and healthy controls (n = 58/MDD, red plots; n = 63/HC, blue plots). The percentage of variation explained by principal coordinates is
marked on the axes. (b) Heatmap of the 54 discriminative operational taxonomic units (OTUs) abundances between depressed subjects and
healthy controls. The taxonomic assignment of each OTU is provided on the right. The increased OTUs in MDD subjects are arranged on the
left, and the decreased OTUs are arranged on the right. As the changed directions of some discriminative OTUs sharing the same taxonomic
assignment at the class level were not identical, there are some colors that are repeated across the same axis. (c) Relative abundances of the
phyla present in samples from MDD patients (left, black bar) and healthy controls (right, green bar). The relative abundances of Actinobacteria
and Bacteroidetes were significantly changed in MDD patients as compared with healthy controls. **Po0.01 by t-test.

Actinomycineae, Coriobacterineae, Lactobacillaceae, Streptococ- relative abundances of Firmicutes between MDD patients and
caceae, Clostridiales incertae sedis XI (Parvimonas), Eubacteria- healthy controls (Figure 2c, Supplementary Figure S5c), change of
ceae, Lachnospiraceae (Anaerostipes, Blautia, Dorea, Firmicutes was still one of hallmark in MDD. This is because some
Lachnospiracea incertae sedis), Ruminococcaceae (Clostridium members of the Firmicutes OTUs (24/56) were increased in MDD
IV) and Erysipelotrichaceae incertae sedis, while a total of 25 patients, while others were decreased (Supplementary Table S5).
OTUs were overrepresented in healthy control subjects and In sum, we observed that MDD was associated with a disturbance
assigned to the families of Bacteroidaceae, Rikenellaceae in the gut microbiome characterized by alterations in specific
(Alistipes), Lachnospiraceae (Coprococcus, Clostridium XlVa, OTUs assigned to the phyla Firmicutes, Actinobacteria and
Lachnospiracea incertae sedis, Roseburia and Faecalibacterium), Bacteroidetes.
Acidaminococcaceae (Phascolarctobacterium), Veillonellaceae
(Megamonas) and Sutterellaceae. Those discriminative OTUs were
mainly assigned to the phyla Firmicutes (45/56, 76.7%), Actino- Transplantation of MDD patient microbiota induces depression-
bacteria (5/56, 10.9%) and Bacteroidetes (3/56, 5.3%). Compared like behaviors in GF recipient mice
with healthy controls, the relative abundances of Actinobacteria To investigate whether changes in gut microbiome contribute to
were increased in MDD subjects, while those of Bacteroidetes the pathogenesis of MDD, FMT experiments were performed.59
were decreased (Figure 2c, Supplementary Figures S5a and b). This approach has been successfully used to determine the
Although there were no significant differences in the overall causative role of gut microbiota in the onset of obesity, colitis and

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Figure 3. Comparative assessment of depression-like behavior in ‘depression microbiota’ and ‘healthy microbiota’ recipients. (a) Forced
swimming test (FST), and (b) tail suspension test (TST): The ‘depression microbiota’ recipients displayed an increased duration of immobility in
the FST and TST compared to ‘healthy microbiota’ recipients (FST: n = 63/depressive group; n = 69/control group; TST: n = 55/depressive group;
n = 63/control group). (c) Open-field test (OFT): There was no difference in total motion distance between ‘depression microbiota’ and ‘healthy
microbiota’ recipients (n = 63/depressive group; n = 69/control group). (d) OFT: Proportion of central motion distance was quantified to assess
anxiety-like behavior. The ‘depression microbiota’ recipients displayed a decreased proportion of central motion distance relative to ‘healthy
microbiota’ recipients. (n = 63/depressive group; n = 69/control group). Data are presented as means ± standard errors of the mean. *Po 0.05,
***P o0.001 by t-test.

type I diabetes.15–19 Here, as previously reported, adult GF mice post FMT. On week 1 post FMT, there were no significant
were colonized with pooled fecal samples randomly derived from differences in OFT, FST and TST between ‘depression microbiota’
five non-medicated MDD patients and five healthy controls and ‘healthy microbiota’ recipient mice (Supplementary Figures
without a priori knowledge of their gut microbial profiles. In the S8a–d). However, on week 2 post FMT, the ‘depression microbiota’
post hoc 16S rRNA gene sequence analysis of these samples, PCoA recipient mice displayed a decreased proportion of center motion
plot of unweighted and weighted UniFrac matrix showed that the distance in the OFT and an increased duration of immobility in the
gut microbial phenotypes of these randomly selected MDD and FST and TST as compared with those of 'healthy control' recipient
healthy control samples were similar to their corresponding mice (Figures 3a and b). However, there were no significant
groups (Supplementary Figure S6). This humanized FMT model differences in total motion distance between the two groups in the
allowed us to: (i) determine whether the depressive phenotypes of OFT (Figure 3c). These findings show that colonization of GF mice
MDD patients were transmissible via their gut microbiomes and (ii) with ‘depression microbiota’ resulted in increased depression-like
comparatively analyze the gut microbial community structure, behaviors as compared with colonization with ‘healthy microbiota’.
metabolism and host-microbial co-metabolism of the ‘depression Given that anxiety is a common symptom among MDD patients,60
microbiota’ and ‘healthy microbiota’ recipient mice. anxiety-like behavior was also tested here. We found that
Initially, the weight of experimental mice was measured weekly. ‘depression microbiota’ recipients displayed a decreased center
We found that the weight of ‘depression microbiota’ recipient mice motion distance in the OFT, which is indicative of anxiety-like
was not significantly different than 'healthy control' recipient mice behavior (Figure 3d). Therefore, in this humanized depressed
during FMT experimentation (Supplementary Figures S7a–c). To model, diagnostic depressive behavior (as measured via the FST
test whether colonization of GF mice with ‘depression microbiota’ and TST) as well as non-diagnostic anxiety behavior (as measured
results in depression-like behaviors, three well-established beha- via the OFT) associated with depression were transmissible via the
vioral tests—OFT, FST and TST—were performed on weeks 1 and 2 gut microbiome.

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Figure 4. 16S rRNA sequencing, metagenomic and metabolomics analyses. (a) Two-dimensional principal coordinate analysis (PCoA) of
unweighted UniFrac distances showed an obvious difference in the microbial community compositions between ‘depression microbiota’
recipient mice and 'healthy control' recipient mice (n = 10 in each group). (b) Relative abundance of operational taxonomic units (OTUs)
responsible for discriminating depressed mice from control mice. The taxonomic assignment of each OTU is provided on the right. The
increased OTUs in depressed mice are arranged on the right, and the decreased OTUs are arranged on the left. (c) Relative abundance of
enzyme commission numbers (E.C.s) responsible for discriminating the fecal microbiomes of depressed and control recipient mice. The key
metabolic pathways of these E.C.s are provided on the right. (d) Fecal and serum metabolites that are associated with carbohydrate
metabolism and amino acid metabolism.

Characterizing gut microbiotic alterations in recipient mice control' recipient mice at 1 week post FMT were analyzed. Two-
To determine whether differences in the gut microbiomes dimensional PCoA analysis of unweighted UniFrac distances
between MDD and healthy control individuals were maintained showed that the gut microbiotic community composition of
in the recipient mice, the microbial communities in the cecum ‘depression microbiota’ recipient mice was different from that of
stools harvested from depressed and control mice were subjected 'healthy control' recipient mice (Supplementary Figure S10). In the
to 16S rRNA gene sequencing at 2 weeks post FMT. The PCoA PCoA analysis of the weighted UniFrac distances, contrasting
plots of weighted and unweighted UniFrac analysis revealed that results were obtained for the two time points, which paralleled the
the key characteristic discriminative gut microbiota observed in observed disparities in behavioral performance. The unweighted
human donors were also represented in recipient mice (Figure 4a UniFrac analysis is relatively sensitive to rarer taxa, whereas the
and Supplementary Figure S9). Using a Random Forests classifier, weighted UniFrac analysis is relatively sensitive to abundances of
40 OTUs responsible for discriminating the gut microbiota in taxa. These findings suggest that the relatively high abundances
‘depression’ and ‘healthy control’ recipient mice were identified of differential gut microbiota detected at 2 weeks post FMT may
(Figure 4b, Supplementary Table S6). These discriminative OTUs be responsible for the observed depression-like behaviors.
were mainly assigned to the phyla Firmicutes (22/39, 56.4%),
Bacteroidetes (13/39, 33.3%) and Actinobacteria (4/39, 10.2%), Metagenomic and metabonomic analyses reveal perturbed
which were similar to the differences observed in the gut metabolic pathways in depressed mice
microbiomes between MDD patients and healthy controls. To characterize the functions encoded by the gut microbiota DNA,
Moreover, disparities in behavioral performance between murine cecum stool samples were collected at the time of killing
'depression' and 'healthy control' recipient mice were observed the mice (week 2 post FMT) and subjected to multiplex
at 1 and 2 weeks post FMT. To uncover the underlying molecular shotgun metagenomic analysis using the Illumina HiSeq2500
basis, the microbial communities of 'depression' and 'healthy (6.3 × 107 ± 7.6 × 106 sequences per sample; Supplementary Table

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S7). These readings were annotated by comparison with the Kyoto DISCUSSION
Encyclopedia of Genes and Genomes (KEGG) database. Linear In this study, we demonstrate that the absence of gut microbiota
discriminant analysis was used to compare the KEGG pathways and induces depression-like behavior in mice and that the composi-
E.C.s representation in the fecal microbiomes of the humanized tion of gut microbiota is significantly altered in MDD patients vis-
depressed and healthy control mice, which yielded 39 E.C.s and 7 a-vis healthy control individuals. More notably, this alteration was
KEGG pathways that were responsible for discriminating the fecal transmissible: colonization of GF mice with ‘depression microbiota’
microbiomes of the two groups (Figure 4c and Supplementary resulted in increased depression-like behaviors as compared with
Figure S11, Supplementary Table S8). To further validate these colonization with ‘healthy microbiota’. Mice harboring ‘depression
findings, unbiased metabolomic analysis of fecal, serum and microbiota’ mainly exhibited disturbances in microbial genes and
hippocampal samples revealed that depressed mice were host metabolites involved in carbohydrate and amino acid
significantly different from control mice (Supplementary Figures metabolism. These results highlight the role of gut microbiota as
S12a–h). The differential metabolites between depressed mice and a potential causative factor in depression through their impact on
healthy controls are presented in Supplementary Tables S9-11, and host metabolism.
the non-differential metabolites are presented in Supplementary There have been numerous previous studies that focused on
Table S12. the effects of the ‘microbiota-gut-brain axis’.20,24,25 Here, we found
The 39 differential E.C.s are primarily associated with carbohy- that the absence of gut microbiota induces decreased immobility
drate and amino acid metabolism. The abundance of gene copies time in the FST. In line with our findings, previous GF murine
associated with several carbohydrate metabolism E.C.s were studies have reported that the absence of gut microbiota
increased in depressed mice relative to control mice (for example, physiologically influences the brain’s serotonergic and neuroen-
pentose phosphate pathway (EC: 4.1.2.4, deoxyriboaldolase; docrine systems,21,62 both of which have been linked to the
EC:2.7.1.45, 2-dehydro-3-deoxygluconokinase), starch and sucrose pathogenesis of depression. These findings are further supported
metabolism (EC:3.2.1.1; alpha-amylase), fructose and mannose by previous studies reporting that probiotic treatment can
metabolism (EC:5.3.1.25; L-fucoseisomerase and amino sugar and ameliorate depression-like behaviors.63–65
nucleotide sugar metabolism (EC:3.2.1.55, alpha-N-arabinofurano- Previously, Naseribafrouei et al.11 reported that some OTUs in
sidase; EC:3.5.99.6, glucosamine-6-phosphate deaminase; the Bacteroidetes phylum are positively correlated with depres-
EC:4.2.1.47, GDP-mannose 4,6-dehydratase)). Consistent with these sion, while other OTUs show a negative correlation with
findings, follow-up untargeted metabolomic analysis of fecal and depression. Moreover, Jiang et al.12 found that Bacteroidetes,
serum samples showed that levels of several key carbohydrate Proteobacteria and Actinobacteria are increased, while Firmicutes
metabolites (for example, α-glucose, β-glucose, fructose and is decreased, in MDD subjects vis-a-vis healthy control individuals.
succinate) were increased in depressed mice (Figure 4d, Here, we found that the relative abundance of Actinobacteria was
Supplementary Tables S9 and S10). In regard to E.C.s involved in increased in MDD subjects, whereas that of Bacteroidetes was
amino acid metabolism, the changes were relatively diverse. We decreased, as compared with healthy controls. Moreover, we
observed increased abundance of E.C.s gene copies involved in found that Firmicutes was also responsible for discriminating MDD
lysine biosynthesis (EC:4.2.1.52, dihydrodipicolinate synthase), from healthy controls, although we found no significant difference
histidine metabolism (EC: 4.3.1.3; histidinase), cysteine and in the overall relative abundance of Firmicutes between the two
methionine metabolism (EC: 3.2.2.9; S-adenosylhomocysteine groups. The characteristic gut microbiota in MDD subjects
nucleosidase), arginine and proline metabolism (EC:4.1.3.16, identified here were significantly different than those found in
4-hydroxy-2-oxoglutarate aldolase; EC:4.1.2.14, 2-dehydro-3- previous studies. This disparity may have resulted from differences
deoxy-phosphogluconate aldolase), and glutamate metabolism in sample sizes, the demographic and clinical characteristics of the
(EC:3.5.1.2, glutaminase) and observed downregulation of E.C.s recruited MDD subjects, and/or the statistical methods used to
gene copies involved in valine, leucine and isoleucine biosynthesis identify the MDD-associated gut microbiota. However, these
(EC:2.2.1.6; acetolactate synthase), phenylalanine, tyrosine and findings consistently demonstrate that MDD is linked to marked
tryptophan biosynthesis (EC:4.2.3.4; 5-dehydroquinate synthase), alterations in gut microbiota composition.
and alanine metabolism (EC:6.3.2.1; pantothenate synthetase). Here, we found that the differences in the gut microbial
Accordingly, some of these alterations were subsequently captured communities observed between MDD subjects and healthy
by untargeted metabolomic analysis of fecal and serum samples: controls was unrelated to confounding factors such as antide-
the levels of glutamate, phenylalanine, phenylalanine biosynthesis- pressant use, sex, age, smoking status or body mass index. Owing
related metabolites (myo-inositol) and alanine-related metabolism to the lack of detailed dietary information, we could not analyze
(aspartate, asparagine, glutathione and uracil) were decreased in the relationship between gut microbial composition and dietary
depressed mice relative to control mice (Figure 4d, Supplementary intake. Given that the lifestyles and dietary habits of individuals
Tables S9 and S10). In addition, consistent with the key pathways within the city of Chongqing are very similar (that is, primarily
identified by the differential E.C.s, KEGG pathway analysis showed dominated by rice consumption accompanied by small amounts
that carbohydrate metabolism (that is, pentose and glucuronate of local meats and vegetables), this potential confounding factor
interconversions as well as starch and sucrose metabolism) and likely does not significantly influence the current findings. Similar
glutamate metabolism were dysregulated in depressed mice to our findings, changes in the gut microbial community from the
(Supplementary Figure S10). first to the third trimester of pregnancy are also unrelated to
Given that the hippocampus is widely recognized as a key brain covariates such as health status, body mass index and dietary
region involved in depression,61 comparative hippocampal meta- intake.16 However, some previous studies have also reported that
bolic profiling of humanized depressed and control mice was also dietary habits and age can influence the gut microbiomes of
performed. In agreement with the fecal and serum findings, healthy individuals.66,67 These discrepancies may result from
hippocampi from depressed mice were characterized by distur- differences in the recruited subjects. Taken together, by recruiting
bances in carbohydrate metabolism (for example, glucose, lactose well-matching subjects, we found that MDD was associated with
and malic acid) and amino acid metabolism (for example, characteristic alterations in the gut microbiome.
phenylalanine, N-acetyl-L-aspartic acid, glycine and leucine) To determine whether gut microbiota have a causative role in
(Supplementary Table S11). In sum, these findings demonstrate the development of depression, a bacterial colonization experi-
that dysbiosis of the gut microbiome may have a causal role in the ment was performed here. We demonstrated that colonization of
development of depression via modulating host metabolism. GF mice with whole gut microbiota sampled from depressed

Molecular Psychiatry (2016), 1 – 11 © 2016 Macmillan Publishers Limited

 Altered gut microbiome induces depression
P Zheng et al
9
individuals led to increased depression-like behaviors, exhibiting as demonstrated here, modifications in microbiota affect behavior,
that whole gut microbiota can serve as a pathogen-like entity. whereas conversely, as documented in our companion paper,
Significantly, the altered phylum-level bacterial taxa of the interventions that affect behavior through different mechanisms,
‘inputted’ human ‘depression microbiota’ (characterized by result in changes in the microbiota.71 On the basis of our results,
alterations in Firmicutes, Actinobacteria and Bacteroidetes) were we propose that novel strategies to target this bidirectional
efficiently captured in the ‘output’ mouse fecal communities of interface of gut microbiota and behavior may represent an
‘humanized’ depressed mice. These findings suggest that the approach for the treatment of MDD that is conceptually novel,
alteration of gut microbiota and the resulting induction of when compared with existing antidepressant treatments.
depression-like behaviors are transmissible. On the basis of these
characteristic changes in the gut microbiome, further research
should identify the specific gut microbiota species that dispro- CONCLUSIONS
portionally contribute to the development of depression, as such We demonstrated in these experiments that gut microbiota can
species may serve as novel targets for depression therapy. physiologically induce depression-like behavior in mice. Moreover,
We also found that the gut microbiota that induced depression- we document characteristic alterations in the gut microbiotic
like behaviors in recipient mice produced observable systemic community of MDD patients and show with state-of-the-art
metabolic alterations. Accordingly, a recent animal study also techniques that gut microbiota can contribute to depression-like
demonstrated that gut microbiota influence autism spectrum behavior through altering host metabolism. These findings
disorder-like behavioral symptoms through impacting host provide an original perspective to uncover the pathologic
metabolism.27 Here, humanized depressed mice were character- mechanism(s) underlying depression as well as revealing the
ized by disturbances in microbial genes and host metabolites need for innovative gut-mediated therapies for depression.
involved in carbohydrate and amino acid metabolism. The
majority of carbohydrate metabolic pathways were enhanced in CONFLICT OF INTEREST
depressed mice relative to control mice, suggesting a higher The authors declare no conflict of interest.
energy demand in depressed mice. This finding may account for
previous studies that have shown a reduction in glucose
metabolism in brains from MDD patients.68 In addition, we found ACKNOWLEDGMENTS
that depressed mice were characterized by obvious disturbances This work was supported by the National Basic Research Program of China (973
in amino acid metabolism. In agreement with these findings, Program, grant no. 2009CB918300 and 2013CB532406) and the National Natural
alterations in peripheral and central amino acid metabolism have Science Foundation of China (Grant nos. 81401140 and 30900456).
been previously identified in MDD patients.69,70 Here, we did not
find any particular metabolite(s) that were obviously perturbed in
depressed mice, suggesting that a systemic metabolic perturba- AUTHOR CONTRIBUTIONS
tion induced by gut microbiota likely contributes to the observed Designed the experiments: PX, HW and JY. Performed the metagenomic
depression-like behavior. analysis: PZ, MLL, ZF and LZ. Performed the metabolomic analysis: CJZ, XJX, BHZ
There were some limitations that should be noted here. First, and PZ. Performed the fecal microbiotic transplantation: ZF, BHZ, MLL, XTZ, XYD
although we were able to identify the disturbed gut microbial and WXL. Analyzed the metagenomic and metabolomic data: JJC, SHF and XJX.
communities of MDD subjects, we did not examine other Collected the clinical samples: DYY, YTY, XYD, XTZ and HQM. Drafted the
neuropsychiatric disorders with similar clinical presentations to manuscript: PX, HW and PZ. Revised the manuscript for intellectual content: JY,
MDD. Further studies that recruit patients with other mental PX and NDM.
disorders (such as bipolar disorder and schizophrenia) are needed
to assess the specificity of altered gut microbiota. Second, all
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Supplementary Information accompanies the paper on the Molecular Psychiatry website (http://www.nature.com/mp)

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