Schizophrenia Research 197 (2018) 470–477

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Schizophrenia Research

journal homepage: www.elsevier.com/locate/schres

Analysis of gut microbiota diversity and auxiliary diagnosis as a

biomarker in patients with schizophrenia: A cross-sectional study
Yang Shen a,1, Jintian Xu b,c,1, Zhiyong Li a, Yichen Huang a, Ye Yuan d, Jixiang Wang d, Meng Zhang d,
Songnian Hu b,c,⁎, Ying Liang a,⁎⁎
a
National Clinical Research Center for Mental Disorders, Peking University Sixth Hospital, Institute of Mental Health, Key Laboratory of Mental Health, Ministry of Health, Peking University, Beijing,
China
b
CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China
c
University of Chinese Academy of Sciences, Beijing, China
d
Beijing Gene Tangram Technology Co. Ltd., China

a r t i c l e i n f o a b s t r a c t

Article history: With the advent of sequencing technology, characterization of schizophrenia with underlying probing of gut
Received 21 July 2017 microbiome can provide abundant clues for diagnosis and prognosis of schizophrenia. In this study, we first com-
Received in revised form 26 December 2017 pared the difference of gut microbiota between schizophrenia patients and healthy controls by 16S rRNA se-
Accepted 1 January 2018
quencing. We further explored whether gut microbiota can be used as a biomarker to assist in the diagnosis of
Available online 17 January 2018
schizophrenia. We restricted inclusion criteria strictly to control confounding bias. Finally, we investigated differ-
Keywords:
ences in fecal microbiota between 64 schizophrenia patients and 53 healthy controls. At the phylum level, we
Schizophrenia found that the abundance of Proteobacteria in the schizophrenia patients was significantly increased. At the
16S rRNA sequencing genus level, the relative abundance of Succinivibrio, Megasphaera, Collinsella, Clostridium, Klebsiella and
Gut microbiota Methanobrevibacter was significantly higher whereas the abundance of Blautia, Coprococcus, Roseburia was de-
Brain-gut axis creased compared to health controls. The receiver operating characteristic curve analysis demonstrated that 12
Biomarker significant microbiota biomarkers were capable of being used as diagnostic factors for distinguishing the schizo-
phrenia cohort from those in the control cohort (AUC = 0.837). We performed PICRUSt analysis and found that
several metabolic pathways differed significantly between healthy controls and schizophrenia patients, including
vitamin B6 and fatty acid. In conclusion, there are some difference of gut microbiota between schizophrenia pa-
tients and healthy controls and the insights from this study could be used to develop microbiota-based diagnosis
for schizophrenia.
© 2018 Published by Elsevier B.V.

1. Introduction
In recent years, the roles of gut microbiota have been an important
topic in the study of mental disease. The brain-gut-axis connects the
central nervous system and gut microbiota by means of nerves, hor-
mones and immunization, which has constructed a bidirectional com-
munication system (Collins et al., 2013). Microbiota plays a regulatory
role via the immune system (Erny et al., 2015), hypothalamic pituitary
adrenal (HPA) axis (Sudo et al., 2004), tryptophan metabolism
(O'Mahony et al., 2015), metabolites produced of bacteria (Tan et al.,
2014) and vague nerve pathway (Bravo et al., 2011). Some studies
⁎ Correspondence to: S. Hu, Beijing Institute of Genomics, Chinese Academy of Sciences,
No.1 Beichen West Road, Chaoyang District, Beijing 100101, China.
⁎⁎ Correspondence to: Y. Liang, National Clinical Research Center for Mental Disorders,
Peking University Sixth Hospital, Institute of Mental Health, Ministry of Health, Peking
University, Haidian District, Huayuanbeilu 51, 100191 Beijing, China.
E-mail addresses: husn@big.ac.cn (S. Hu), liangying1980@bjmu.edu.cn (Y. Liang).
1
These authors contributed equally to this work.
have shown that the brain-gut-axis plays an important role in the path-
ogenesis of mental disorders. In some studies of autism, a higher abun-
dance of bacteria was found in the genus Clostridium in fecal sample
(Finegold et al., 2002; Parracho et al., 2005; Song et al., 2004) and also
exhibited decreased Bacteroidetes/Firmicutes ratio (Tomova et al.,
2015). Beyond that, the elevation of Lactobacillus and Desulfovibrio is
related to the severity of autism (Tomova et al., 2015). A recent study
of major depression disorder (MDD) also found a relative abundance
of Firmicutes, Actinobacteria, and Bacteroidetes. In addition, this study
showed that fecal transplantation of germ-free mice with microbiota
from MDD patients led to depressive-like behavioral alteration (Zheng
et al., 2016).Similarly, more and more studies in this field suggested
that a link between dietary and gastrointestinal system and Alzheimer's
disease, which is worth investigating (Gubiani et al., 2017). However,
there is still lack of studies on schizophrenia.
Although the etiology of schizophrenia is still unknown, some clini-
cal studies found that the immune function of patients with schizophre-
nia was increased and they were in the state of inflammation (Castro-

https://doi.org/10.1016/j.schres.2018.01.002
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 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477
Nallar et al., 2015; Song et al., 2013). Moreover, a number of studies
have shown that patients with schizophrenia have intestinal inflamma-
tion and gut microbial disorders (Fond et al., 2015; Severance et al.,
2012; Severance et al., 2013; Sherwin et al., 2016; Yolken et al., 2015).
Douglas-Escobar et al. (2013) and Sudo et al. (2004) had shown that
gut microbes can also control a variety of the expression of neurotrophic
factors, such as brain-derived neurotropic factor (BDNF),
synaptophysin, and PSD-95, which can affect the neural development
and plasticity of the brain. In addition, the levels of markers used to
study bacterial translocation were elevated in acute and chronic schizo-
phrenics (Severance et al., 2012; Severance et al., 2013). The emergence
of research evidence above has encouraged scientists to focus on the gut
microbiota in studies of schizophrenia.
However, current researches on the relationship between gut micro-
biota and schizophrenia remain limited. Schwarz et al. (2017) have
found that the number of certain bacteria has changed significantly by
comparing the gut microbiota between patients with first episode psy-
chosis and health controls, which was correlated with the severity of
psychotic syndromes and the patients' response to treatment. By se-
quencing the oropharynxgeal microbes, previous studies sequenced
the oropharynxgeal microbes and also found the difference between
the schizophrenia patients and healthy controls (Castro-Nallar et al.,
2015; Yolken et al., 2015). However, the size of the samples in these
studies was too small and their subjects included other mental disorders
such as schizoaffective disorder, which may affect the final results.
Meanwhile, the gut microbiota was susceptible to many factors, includ-
ing diet, exercise, metabolism and antipsychotic drugs. In our study, we
only focused on the patients with schizophrenia, supplemented the
sample size, and controlled diet, exercise, metabolism and drug use to
eliminated possible bias. By comparing the differences of gut microbiota
between schizophrenia patients and healthy controls, we preliminarily
explore the correlation between them.
2. Materials and methods
2.1. Participants
In our study, we referred to the human microbiome project consor-
tium standard to screen healthy controls (Human Microbiome Project,
2012). The inclusion criteria was as follows: 1) the Han nationality,
local residents of Huludao area in China, no special religious beliefs;
2) age 18–65 years old; 3) BMI 18–35 kg/m2; 4) absence of any chronic
disease or disease that may affect the stability of gut microbiota, such as
hypertension, diabetes, immunodeficiency, autoimmune diseases, can-
cer, inflammatory bowel disease, diarrhea in the last 3 months; 5) ab-
sence of any specific of drug use for the latest 6 months, including
antibiotics, glucocorticoids, cytokines, large doses of probiotics and bio-
logical agents; 6) absence of gastroscopy, colonoscopy or gastrointesti-
nal barium meal;7) absence of any major surgery with gastrointestinal
tract within 5 years; 8) absence of activity limitation caused by major
physical disease or psychiatric symptoms; 9) absence of significant
changing in dietary habits and middle or high doses of alcohol abuse
or dependence.
In addition, we draw on the experience of criterion in previous re-
search by Dickerson et al. (2003) and adjusted it according to the Chi-
nese situation. The patients were examined and diagnosed according
to the ICD-10 by two trained psychiatric physicians. We eliminated
the diagnosis of schizoaffective disorder and other schizophrenia spec-
trum disorders. Supplementary criteria was as follows: 1) patients
with schizophrenia were diagnosed according to ICD-10 and received
antipsychotic treatment in hospital or outpatient clinic; 2) illness dura-
tion ≤10 years and received antipsychotic drugs treatment N6 months;
3) psychiatric symptoms were steady N3 months, and the Positive and
Negative Syndrome Scale (PANSS) evaluated the rate of change ≤20%
and the total score of PANSS ≤60.
471
2.2. Clinical assessment
All participants had signed the informed consent with their agree-
ment. The sample collection and the protocol of analysis were approved
by Liaoning Province Demobilize Soldiers Hospital according to the Hel-
sinki declaration. A questionnaire was conducted among all subjects to
collect general information, including age, gender, race, height, weight,
anamnesis, history of taking medicine, history of smoking and drinking.
2.3. 16S rRNA amplification of V3–V4 region and illumina sequencing
The fresh fecal samples were obtained from 117 individuals and all
of the samples were stored at −80 °C until DNA extraction. A total of
200 mg of each fecal sample was used for DNA extraction, using
PowerSoil DNA kit (MoBio, USA) according to the manufacturer's in-
structions. The KAPA HiFiHotStartReadyMix (KAPA, USA) was used to
amplify the 16S rRNA (V3–V4) gene tags. Each DNA sample of the bac-
terial 16S rRNA gene was generated by amplified with primers 341F
(GGACTACHVGGGTWTCTAAT) and 805R (ACTCCTACGGGAGGCAGCA
G). Primers contained Illumina adapters and a unique 8-nucleotide
barcode. The PCR conditions included an initial denaturation at 95 °C
for 5 min, 20 cycles of denaturation at 98 °C for 20s, annealing at 58 °C
for 30 s, and elongation at 72 °C for 30 s, and a final extension at 72 °C
for 5 min. PCR amplifications were performed in triplicate using 50 μL
reactions, 10 pmol of forward and reverse primers, and 100 ng of tem-
plate. Amplicons were analyzed on 1.5% agarose gel electrophoresis
and bands of the desired size were purified using the QIAquick Gel Ex-
traction Kit (QIAGEN, Germany). The products were submitted to the
next-generation sequencing laboratory of Novogene Bioinformatics In-
stitute, Beijing, China, using paired-end sequencing with an
IlluminaHiSeq 2500 platform, and 250 bp were sequenced from each
end.
2.4. Bioinformatics analysis
Raw sequence data were processed and analyzed using QIIME
(Quantitative Insights Into Microbial Ecology, Version 1.9.1) software
(Caporaso et al., 2010). Forward and reverse reads for each individual
sample were demultiplexed, joined, and quality filtered. The reads
were truncated at any site receiving an average quality score b 20 over
a 3 bp sliding window, abandon the truncated reads that were shorter
than 75% of raw reads. Sequences contain ambiguous characters, or con-
taining more than two nucleotide mismatches in primer matching were
removed. Chimeric sequences were identified and removed from the
dataset using usearch61 method with reference data of the ‘gold’ set
in http://sourceforge.net/projects/microbiomeutil/files/, version 2011-
11-02(PMID: 21700674). The “open-reference” QIIME protocol was
used with the UCLUST method other default parameters to select oper-
ational taxonomix units (OTUs). Sequences with at least 97% similarity
were clustered together, and representative sequence from each cluster
was used to identify bacterial taxa from the Greengenes database as of
13 August 2013 (DeSantis et al., 2006). OTUs containing fewer than 2 se-
quences or with overall relative abundance b0.00005 were excluded
from further analysis. Because we obtained a variable number of se-
quences per sample ranging from 11,286 to 61,443, the sequence data
were rarefied to 10,000 sequences per sample to account for variations
in sequencing depth. QIIME was used to calculate alpha indices and beta
diversity.
Linear discriminant analysis (LDA) effect size (LEfSe) was used for
the identification of the different markers, an alpha = 0.05 was used
in Wilcoxon rank sum test, and the log value for the LDA analysis was
set to be b2.0 (Segata et al., 2011). To obtain insight into the possible
functional pathway that differ between schizophrenia and healthy co-
horts, we used PICRUSt to calculate contributions of various OTUs to
known biological pathways based on KEGG orthology groups (KOs)
472 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477
using Kyoto Encyclopedia of Genes and Genomes (KEGG) databases
(Langille et al., 2013).
Statistical analysis was performed using the R-3.3.1 and Statistical
Analysis of Metagenomic Profiles software (Parks et al., 2014). For con-
tinuous variables, independent t-test, Welch's t-test, White's nonpara-
metric t-test were applied. For categorical varibles between groups,
using either Pearson chi-square or Fisher's exact test, depending on as-
sumption validity. Principal coordinate analysis (PCoA) based on
unweightedUniFrac distance matrix was used for visualizing sample re-
lationships and ANOSIM was used to test significant difference in micro-
bial community composition. All tests of significance were two sided,
and p b .05, or corrected p b .05, was considered statistically significant.
The machine learning algorithm, Boruta (Kursa and Rudnicki, 2010)
variable selection was applied to select the most discriminatory taxa
based on the importance values of microbiota markers identified by
LEfSe. The importance value of a taxa was calculated based on the loss
of accuracy by random permutation of the abundance of taxon. And an-
other method, random forests (Liaw and Wiener, 2002), was used to de-
velop classifier and predict schizophrenia from healthy cohort based on
the important microbiota. The microbiota markers proportion data
served as input data. Bootstrapping (N = 100) was used to grow classi-
fication trees in the forest using the training dataset. About two third of
the samples was randomly selected to grow a classification tree and rest
one third of the samples was used for the prediction. The predictive
model was established with default parameters of the R implementa-
tion of the algorithms (R packages ‘Boruta’ and ‘random forest’). The
classification performance of each bootstrap was calculated and the re-
ceiver operating characteristic curve was plotted of and the area under
curve was calculated using R package pROC (Robin et al., 2011).
The raw sequence data reported in this article have been deposited
in the Genome Sequence Archive (Wang et al., 2017) in the BIG Data
Center (BIG Data Center Members, 2017), Beijing Institute of Genomics
(BIG), Chinese Academy of Sciences, under accession numbers
CRA000653 that are publicly accessible at http://bigd.big.ac.cn/gsa.
3. Result
3.1. Clinical data
According to the inclusion and exclusion criteria, a total of 64 schizo-
phrenia patients and 53 healthy controls were recruited and the male to
female ratio of cases was 36:28 and 35:18, respectively. There was no
statistical difference in age, body mass index (BMI), sex ratio, tobacco
used and alcohol intake between two groups (p N .05, Table 1).
3.2. Sequencing data
We obtained 6,975,476 raw sequences from the 117 samples, range
from 21,189 to 107,167. After quality filtering and removing the chime-
ric sequences, we obtained 3,863,613 high quality reads for further anal-
ysis of bacterial composition, with a mean of 33,022.33 reads (ranging
from 11,286 to 61,443, Supplementary Table S1). The mean read length
was 453 bp (ranging from 330 to 474). The value of good's coverage es-
timator was 99.79%.
Table 1
Demographic characteristics of the participants of schizophrenia and health controls.
Values are shown as mean ± SD or ratio.
Characteristic Healthy (n = 53) Schizophrenia (n = 64) p-Value
Age 39 ± 14 42 ± 11 0.169
BMI (kg/m2) 23.14 ± 2.8 23.49 ± 3.8 0.579
Sex ratio (M/F) 35/18 36/28 0.281
Tobacco intake (%) 22.6 18.8 0.604
Alcohol intake (%) 5.7 1.6 0.225
BMI, body mass index.
To characterize the richness and diversity of bacterial community,
we calculated alpha indices for each sample (Supplementary
Table S2). As shown in Table 2, the richness and diversity estimators
in the two cohorts were not significantly different. To measure the ex-
tent of the similarity of fecal microbial communities, beta diversity
was calculated using unweighted UniFrac distances and principal coor-
dinate analysis, in which we found statistically different between the
two groups (R = 0.044, p = .016; Fig. 1). Additionally, the fecal micro-
biota of healthy cohort displayed significantly tighter clustering com-
pared to the schizophrenia patients with average unweighted Unifrac
distances of 0.38 ± 0.09 vs. 0.42 ± 0.08, p b .001, respectively (Supple-
mentary Fig. S1).
3.3. Bacterial taxonomic composition of healthy and schizophrenia cohorts
The predominant bacteria in the healthy cohort included
Bacteroidetes, Firmicutes, Proteobacteria and Actinobacteria (Fig. 2A),
while the schizophrenia cohort was dominated by Bacteroidetes,
Firmicutes, Proteobacteria, Actinobacteria and Fusobacteria (Fig. 2B).
When the relative abundance of bacterial phylum was compared,
Proteobacteria was found more abundant in the schizophrenia than
those from the healthy cohort (7.59% vs. 4.54%, p = .004, respectively,
Welch's t-test).
At the genus level, healthy controls were mainly assigned to
Bacteroides, Prevotella, Faecalibacterium, Sutterella, Ruminococcus, and
Parabacteroides (Fig. 3A). The most abundant genus was also Bacteroides
in Schizophrenia patients, followed by Prevotella, Faecalibacterium, and
Succinivibrio (Fig. 3B). The heatmap of genera relative abundance was
showed in the Supplementary Fig. S2. Genera with different relative
abundances between the two cohorts are listed in Fig. 3B (p b .05). Com-
pared to healthy cohort, the relative abundance of Succinivibrio,
Megasphaera, Collinsella, Clostridium, Klebsiella and Methanobrevibacter
were significant higher in the schizophrenia cohort. However, Blautia,
Coprococcus and Roseburiawere higher in the healthy cohorts. Hierarchi-
cal clustering based on the relative abundance different genera demon-
strated that the samples from patients generally clustered together
(Fig. 3C).
The significantly different bacterial species are listed in Supplemen-
tary Fig. S3. Those in the schizophrenia cohort had significantly higher
Collinsellaaerofaciens and Bacteroidesfragilis. In contrast, they also had a
lower relative abundance of Roseburiafaecis, Blautiaproducta and
Collinsellaplebeius.
3.4. Identification of biomarkers and diagnostic factors determination
We used LEfSe for the quantitative analysis of biomarkers within dif-
ferent cohorts. A total of 42 features had significantly different abun-
dance between healthy and schizophrenia patients (Fig. 4B). At genus
level, fecal microbiota of schizophrenia cohort was differently enriched
with genera Prevotella, Succinivibrio, Fusobacterium, Lactobacillus,
Megasphaera, Acidaminococcus, Citrobacter, Phascolarctobacterium and
Desulfovibrio, whereas healthy control cohort was enriched with Strep-
tococcus, Coprococcus, Roseburia and Blautia. We also observed that
Table 2
Richness and diversity estimators in the healthy and schizophrenia cohorts.
Parameter Healthy Schizophrenia p-Value
Number of reads 34,990 ± 10,048 31,392 ± 13,016 0.08
PD_whole_tree 13.31 ± 2.63 13.13 ± 2.55 0.77
Goods_coverage 99.83 ± 0.06 99.76 ± 0.16 0.10
Observed_otu 341.77 ± 81.43 320.39 ± 74.97 0.14
Shannon 5.45 ± 0.89 5.32 ± 0.77 0.36
Simpson 0.92 ± 0.06 0.93 ± 0.04 0.64
ACE 384.83 ± 80.95 367.55 ± 71.06 0.15
Chao1 394.02 ± 81.25 373.91 ± 71.25 0.09
 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477
Fig. 1. UnweightedUniFrac principal coordinates analysis (PCoA) plot camparing sample
distribution between the two cohorts. Red and green dots represent healthy controls
and schizophrenia patients, respectively.
phylum Proteobacteria and Fusobacteria were enriched in schizophrenia
patients while Firmicutes in healthy control subjects (Fig. 4B).
To identify biomarkers that contributed significantly to the predic-
tion performance, we applied the Boruta feature selection algorithm,
which is built around random forest and select features that have signif-
icantly more classification power than random permuted features. The
Fig. 2. Microbial composition at phylum level. (A–B) indicate the most abundant phyla detected in the healthy and schizophrenia cohorts. Compared to healthy controls, schizophrenia
patients had a significantly higher abundance of Proteobacteria.
473
important biomarkers include Gammaproteobacteria at class level,
Enterobacteriales at order level, Alcaligenaceae, Enterobacteriaceae and
Lachnospiraceae at family level, Acidaminococcus, Phascolarctobacterium,
Blautia, Desulfovibrio and Megasphaera at genus level and plebeius,
fragilis at species level (The importance of taxa was shown in Supple-
mentary Fig. S4). Then we applied random forest method to develop
classifier based on the 12 microbiota biomarkers. We achieved a mean
classification error of 0.24, and mean of auc is 0.837 (Supplementary
Table S3). Therefore, this result demonstrated that these features were
capable of being used as diagnostic factors for distinguishing those in
the schizophrenia cohort from those in the control cohort.
3.5. Functional properties predicted by PICRUSt
We performed PICRUSt analysis to predict the genetic potentials of
the fecal microbiota metagenomes based on 16S rRNA sequences.
PICRUSt assignment of predicted metagenome content to Level 3 KOs
and identified 63 different functional pathways (Supplementary
Table S4, FDR b 0.05). Interestingly, several metabolic pathways differed
significantly between healthy and schizophrenia cohorts, including vi-
tamin B6, fatty acid, starch and sucrose, tryptophan, cysteine, methio-
nine and linoleic acid metabolism, as well as the degradation of some
xenobiotics. We next analyzed the relationship between genera altered
in schizophrenia patients and differentially metabolic pathways. Genera
presented at least 75% samples in both patients and controls were con-
sidered and found to be related to many of metabolic pathways (Fig. 5).
For example, Blautia, Coprococcus, Roseburia were negatively associated
with Vitamin B6, Taurine and Hypotaurine metabolic pathway, and pos-
itively associated with Methane metabolic pathway.
4. Discussion
In our study, firstly we found the difference of gut microbiome be-
tween the patients with schizophrenia and health controls by 16S se-
quencing. The gut microbiota of normal people mainly consists of four
major clades: Bacteroidetes, Firmicutes, Proteobacteria, and
Actinobacteria, that is consistent with previous findings (De Filippo
et al., 2010; Human Microbiome Project, 2012; Nam et al., 2011;
Zhang et al., 2015). It accounts for nearly 99% of the total bacterial
count in the intestinal tract. The abundance of Proteobacteria was higher
in schizophrenia patients than healthy controls. Furthermore, the abun-
dance of Succinivibrio at the genus level was much higher in
474 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477

Fig. 3. Microbial composition at genus level. (A) Summary of bacterial genera detected in the two cohorts. (B) Genus-level bacteria that were significantly different between the two
cohorts. Welch's t-test was used to compare the differences in the relative abundance of bacterial genera between the healthy controls and schizophrenia patients (p b .05).
(C) Heatmap based on the abundance of different genera. Red and green indicate low and high abundance, respectively. Hierarchical clustering (manhattan distance, complete linkage)
shows that schizophrenia samples tend to cluster together.

Fig. 4. Differently abundant taxa identified using LEfSe analysis. A. LEfSecladogram showed the most differentially abundant taxa between the two cohorts. Taxa enriched for healthy in
red; schizophrenia enriched taxa in green. The brightness of each dot is proportional to its effect size. B. Visualization of only taxa meeting an LDA threshold N2.
 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477
Fig. 5. Heatmap of correlation between the relative abundances of the alter genera and the differentially relative abundances metabolism pathway. Colors indicate the Pearson correlation
coefficients (*P b .05, Benjamini-Hochberg FDR false discovery rate correction).
schizophrenia than healthy controls in our study, which belong to the
Proteobacteria. The majority of the other genera were found to belong
to the Firmicutes, including Blautia, Coprococcus, Roseburia, Clostridium
and Megasphaera. In the Castro-Nallar et al. (2015) study, it was also
found that the proportion of Firmicutes was higher in schizophrenia,
while the Proteobacteria was not significantly different between the
two groups. It may suggest that there may be a wide range of
Firmicutes disorders in the digestive tract of patients with
schizophrenia, while the Proteobacteria imbalance may be more
prominent in the gut.
The short chain fatty acids (SCFA) acetate, propionate, and butyrate
are the primary metabolic products by gut microbes (Wong et al., 2006).
In our study, we found the abundance of Blautia, Coprococcus and
Roseburia were decreased in schizophrenia at the genus level. These
bacteria reductions were often associated with a reduction in SCFAs
(Li et al., 2017). In addition, the abundance of Collinsella was elevated,
which had been shown to produce proinflammatory cytokines
interleukin-17a and to alter intestinal permeability in previous studies
in arthritis (Chen et al., 2016). SCFAs can improve the intestinal barrier
function (Peng et al., 2009) and regulate a variety of immune and epige-
netic pathways (Meijer et al., 2010) such as interleukin-6 and tumor ne-
crosis factor released by macrophages (Kim et al., 2014). Except for the
bacterial antigens and their metabolic products, some special food anti-
gens can elevate the levels of human immune factors (Cascella et al.,
2011; Eaton et al., 2006; Okusaga et al., 2013). A previous Meta-
analysis including 40 studies showed that the inflammatory factors in
the serum of schizophrenia patients were elevated (Miller et al.,
2011). Inflammatory factors in the blood arrived at intracranial accord-
ing to the blood circulation system through the blood-brain barrier di-
rectly or interacted with astrocyte into the brain (Banks et al., 1995;
Verkhratsky et al., 2016), and caused inflammatory effect by activating
the microglia (Norden et al., 2016). In addition, we found varieties of
metabolic pathways related to the differential gut microbiota in the pre-
diction of gut microbiota function, and the influence on vitamin B6 me-
tabolism pathway was significant. Moreover, a study showed that folic
acid (vitamin B9) was associated with the occurrence of negative symp-
toms inschizophrenia, which vitamin B was found to be related to
schizophrenia before (Ellingrod et al., 2008). Vitamin B6 is cofactor of
N150 enzymes (Percudani and Peracchi, 2009), participated in many
pathways of substance metabolism, such as the metabolic pathway of
homocysteine (Kim and Moon, 2011), which may affect mental state.
Severe deficiencies of vitamin B6 can lead to psychomotor, cognitive
and mood deficits (Selhub et al., 2009). Therefore, it is necessry to fur-
ther explore that whether supplement vitamin B6 could improve psy-
chiatric symptoms.
475
So far, there are no validated laboratory tests, biomarkers or a panel
of combined tests to assess diagnosis, prognosis or the prediction of the
treatment response of schizophrenia (Perkovic et al., 2017). However,
we can distinguish the two groups based on the 12 characteristic
quantity of bacteria in our study, which was selected by the Boruta
feature selection algorithm (AUC reached 0.837). It is of high
diagnostic efficacy that might be established according to this
identification method. In the future, we might be able to assist
psychiatric physicians to diagnose the schizophrenia by measuring
gut microbiota.
There are several limitations in our study. It is just a cross-sectional
study of people with schizophrenia in Chinese Han nationality, the sam-
ple size is too small. And we did not completely eliminate the effect of
antipsychotics on the gut microbiota. So in subsequent studies, we
could consider increasing the sample size and select multi-ethnic of pa-
tients with the untreatment of first episode schizophrenia for longitudi-
nal follow-up studies to observe the dynamic changes of gut microbiota.
However, our advantage is by comparing the differences in the gut mi-
crobiota between schizophrenia and health people found the potential
of the gut microbiota to cause the disease and auxiliary diagnosis as a
biomarker.
5. Conclusion
There are some differences of gut microbiota between schizophrenia
patients and healthy controls and the insights from this study could be
used to develop microbiota-based diagnosis for schizophrenia.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.schres.2018.01.002.
Role of the funding source
None.
Author contributions
Yang Shen, Jintian Xu, Songnian Hu and Ying Liang designed the
study and wrote the protocol. Zhiyong Li, Ye Yuan and Jixiang Wang
managed the literature searches and analyses. Yichen Huang and
Meng Zhang undertook the statistical analysis, and Yang Shen and
Jintian Xu wrote the first draft of the manuscript. All authors contrib-
uted to and have approved the final manuscript.
476 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477
Conflict of interests
The authors declare no conflicts of interest in this work.
Acknowledgements
We thank all the subjects who took part in this study.
References
Banks, W.A., Kastin, A.J., Broadwell, R.D., 1995. Passage of cytokines across the blood-brain
barrier. Neuroimmunomodulation 2 (4), 241–248.
BIG Data Center Members, 2017 Jan 4. The BIG Data Center: from deposition to integra-
tion to translation. Nucleic Acids Res. 45 (D1):D18–D24. https://doi.org/10.1093/
nar/gkw1060.
Bravo, J.A., Forsythe, P., Chew, M.V., Escaravage, E., Savignac, H.M., Dinan, T.G.,
Bienenstock, J., Cryan, J.F., 2011. Ingestion of Lactobacillus strain regulates emotional
behavior and central GABA receptor expression in a mouse via the vagus nerve.
Proc. Natl. Acad. Sci. U. S. A. 108 (38), 16050–16055.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K.,
Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T., Knights,
D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D., Pirrung, M.,
Reeder, J., Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A., Widmann, J., Yatsunenko, T.,
Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-throughput community
sequencing data. Nat. Methods 7 (5), 335–336.
Cascella, N.G., Kryszak, D., Bhatti, B., Gregory, P., Kelly, D.L., Mc Evoy, J.P., Fasano, A., Eaton,
W.W., 2011. Prevalence of celiac disease and gluten sensitivity in the United States
clinical antipsychotic trials of intervention effectiveness study population. Schizophr.
Bull. 37 (1), 94–100.
Castro-Nallar, E., Bendall, M.L., Perez-Losada, M., Sabuncyan, S., Severance, E.G., Dickerson,
F.B., Schroeder, J.R., Yolken, R.H., Crandall, K.A., 2015. Composition, taxonomy and
functional diversity of the oropharynx microbiome in individuals with schizophrenia
and controls. PeerJ 3, e1140.
Chen, J., Wright, K., Davis, J.M., Jeraldo, P., Marietta, E.V., Murray, J., Nelson, H., Matteson,
E.L., Taneja, V., 2016. An expansion of rare lineage intestinal microbes characterizes
rheumatoid arthritis. Genome Med. 8 (1), 43.
Collins, S.M., Kassam, Z., Bercik, P., 2013. The adoptive transfer of behavioral phenotype
via the intestinal microbiota: experimental evidence and clinical implications. Curr.
Opin. Microbiol. 16 (3), 240–245.
De Filippo, C., Cavalieri, D., Di Paola, M., Ramazzotti, M., Poullet, J.B., Massart, S., Collini, S.,
Pieraccini, G., Lionetti, P., 2010. Impact of diet in shaping gut microbiota revealed by a
comparative study in children from Europe and rural Africa. Proc. Natl. Acad. Sci. U. S.
A. 107 (33), 14691–14696.
DeSantis, T.Z., Hugenholtz, P., Larsen, N., Rojas, M., Brodie, E.L., Keller, K., Huber, T., Dalevi,
D., Hu, P., Andersen, G.L., 2006. Greengenes, a chimera-checked 16S rRNA gene data-
base and workbench compatible with ARB. Appl. Environ. Microbiol. 72 (7),
5069–5072.
Dickerson, F.B., Boronow, J.J., Stallings, C., Origoni, A.E., Ruslanova, I., Yolken, R.H., 2003.
Association of serum antibodies to herpes simplex virus 1 with cognitive deficits in
individuals with schizophrenia. Arch. Gen. Psychiatry 60 (5), 466–472.
Douglas-Escobar, M., Elliott, E., Neu, J., 2013. Effect of intestinal microbial ecology on the
developing brain. JAMA Pediatr. 167 (4), 374–379.
Eaton, W.W., Byrne, M., Ewald, H., Mors, O., Chen, C.Y., Agerbo, E., Mortensen, P.B., 2006.
Association of schizophrenia and autoimmune diseases: linkage of Danish national
registers. Am. J. Psychiatry 163 (3), 521–528.
Ellingrod, V.L., Miller, D.D., Taylor, S.F., Moline, J., Holman, T., Kerr, J., 2008. Metabolic syn-
drome and insulin resistance in schizophrenia patients receiving antipsychotics ge-
notyped for the methylenetetrahydrofolate reductase (MTHFR) 677C/T and 1298A/
C variants. Schizophr. Res. 98 (1–3), 47–54.
Erny, D., Hrabe de Angelis, A.L., Jaitin, D., Wieghofer, P., Staszewski, O., David, E., Keren-
Shaul, H., Mahlakoiv, T., Jakobshagen, K., Buch, T., Schwierzeck, V., Utermohlen, O.,
Chun, E., Garrett, W.S., McCoy, K.D., Diefenbach, A., Staeheli, P., Stecher, B., Amit, I.,
Prinz, M., 2015. Host microbiota constantly control maturation and function of mi-
croglia in the CNS. Nat. Neurosci. 18 (7), 965–977.
Finegold, S.M., Molitoris, D., Song, Y., Liu, C., Vaisanen, M.L., Bolte, E., McTeague, M.,
Sandler, R., Wexler, H., Marlowe, E.M., Collins, M.D., Lawson, P.A., Summanen, P.,
Baysallar, M., Tomzynski, T.J., Read, E., Johnson, E., Rolfe, R., Nasir, P., Shah, H.,
Haake, D.A., Manning, P., Kaul, A., 2002. Gastrointestinal microflora studies in late-
onset autism. Clin. Infect. Dis. 35 (Suppl. 1), S6–S16.
Fond, G., Boukouaci, W., Chevalier, G., Regnault, A., Eberl, G., Hamdani, N., Dickerson, F.,
Macgregor, A., Boyer, L., Dargel, A., Oliveira, J., Tamouza, R., Leboyer, M., 2015. The
“psychomicrobiotic”: targeting microbiota in major psychiatric disorders: a system-
atic review. Pathol. Biol. 63 (1), 35–42.
Gubiani, D., Fabbretti, E., Cestnik, B., Lavrač, N., Urbančič, T., 2017. Outlier based literature
exploration for cross-domain linking of Alzheimer's disease and gut microbiota. Ex-
pert Syst. Appl. 85, 386–396.
Human Microbiome Project, C, 2012. Structure, function and diversity of the healthy
human microbiome. Nature 486 (7402), 207–214.
Kim, T.H., Moon, S.W., 2011. Serum homocysteine and folate levels in Korean schizo-
phrenic patients. Psychiatry Investig. 8 (2), 134–140.
Kim, C.H., Park, J., Kim, M., 2014. Gut microbiota-derived short-chain fatty acids, T cells,
and inflammation. Immune Network 14 (6), 277–288.
Kursa, M.B., Rudnicki, W.R., 2010. Feature selection with the Boruta package. J. Stat. Softw.
36 (11), 1–13.
Langille, M.G., Zaneveld, J., Caporaso, J.G., McDonald, D., Knights, D., Reyes, J.A., Clemente,
J.C., Burkepile, D.E., Vega Thurber, R.L., Knight, R., Beiko, R.G., Huttenhower, C., 2013.
Predictive functional profiling of microbial communities using 16S rRNA marker gene
sequences. Nat. Biotechnol. 31 (9), 814–821.
Li, J., Zhao, F., Wang, Y., Chen, J., Tao, J., Tian, G., Wu, S., Liu, W., Cui, Q., Geng, B., Zhang, W.,
Weldon, R., Auguste, K., Yang, L., Liu, X., Chen, L., Yang, X., Zhu, B., Cai, J., 2017. Gut micro-
biota dysbiosis contributes to the development of hypertension. Microbiome 5 (1), 14.
Liaw, A., Wiener, M., 2002. Classification and regression by random forest. R News 2 (3),
18–22.
Meijer, K., de Vos, P., Priebe, M.G., 2010. Butyrate and other short-chain fatty acids as
modulators of immunity: what relevance for health? Curr. Opin. Clin. Nutr. Metab.
Care 13 (6), 715–721.
Miller, B.J., Buckley, P., Seabolt, W., Mellor, A., Kirkpatrick, B., 2011. Meta-analysis of cyto-
kine alterations in schizophrenia: clinical status and antipsychotic effects. Biol. Psy-
chiatry 70 (7), 663–671.
Nam, Y.D., Jung, M.J., Roh, S.W., Kim, M.S., Bae, J.W., 2011. Comparative analysis of Korean
human gut microbiota by barcoded pyrosequencing. PLoS One 6 (7), e22109.
Norden, D.M., Trojanowski, P.J., Villanueva, E., Navarro, E., Godbout, J.P., 2016. Sequential
activation of microglia and astrocyte cytokine expression precedes increased Iba-1 or
GFAP immunoreactivity following systemic immune challenge. Glia 64 (2), 300–316.
Okusaga, O., Yolken, R.H., Langenberg, P., Sleemi, A., Kelly, D.L., Vaswani, D., Giegling, I.,
Hartmann, A.M., Konte, B., Friedl, M., Mohyuddin, F., Groer, M.W., Rujescu, D.,
Postolache, T.T., 2013. Elevated gliadin antibody levels in individuals with schizo-
phrenia. World J. Biol. Psychiatry 14 (7), 509–515.
O'Mahony, S.M., Clarke, G., Borre, Y.E., Dinan, T.G., Cryan, J.F., 2015. Serotonin,
tryptophan metabolism and the brain-gut-microbiome axis. Behav. Brain Res.
277, 32–48.
Parks, D.H., Tyson, G.W., Hugenholtz, P., Beiko, R.G., 2014. STAMP: statistical analysis of
taxonomic and functional profiles. Bioinformatics 30 (21), 3123–3124.
Parracho, H.M., Bingham, M.O., Gibson, G.R., McCartney, A.L., 2005. Differences between
the gut microflora of children with autistic spectrum disorders and that of healthy
children. J. Med. Microbiol. 54 (Pt 10), 987–991.
Peng, L., Li, Z.R., Green, R.S., Holzman, I.R., Lin, J., 2009. Butyrate enhances the intestinal
barrier by facilitating tight junction assembly via activation of AMP-activated protein
kinase in Caco-2 cell monolayers. J. Nutr. 139 (9), 1619–1625.
Percudani, R., Peracchi, A., 2009. The B6 database: a tool for the description and classifica-
tion of vitamin B6-dependent enzymatic activities and of the corresponding protein
families. BMC Bioinf. 10, 273.
Perkovic, M.N., Erjavec, G.N., Strac, D.S., Uzun, S., Kozumplik, O., Pivac, N., 2017.
Theranostic biomarkers for schizophrenia. Int. J. Mol. Sci. 18 (4).
Robin, X., Turck, N., Hainard, A., Tiberti, N., Lisacek, F., Sanchez, J.C., Muller, M., 2011.
pROC: an open-source package for R and S+ to analyze and compare ROC curves.
BMC Bioinf. 12, 77.
Schwarz, E., Maukonen, J., Hyytiainen, T., Kieseppa, T., Oresic, M., Sabunciyan, S., Mantere,
O., Saarela, M., Yolken, R., Suvisaari, J., 2017 Apr 22. Analysis of microbiota in first
episode psychosis identifies preliminary associations with symptom severity and
treatment response. Schizophr Res. https://doi.org/10.1016/j.schres.2017.04.017
(pii: S0920-9964(17)30204-9).
Segata, N., Izard, J., Waldron, L., Gevers, D., Miropolsky, L., Garrett, W.S.,
Huttenhower, C., 2011. Metagenomic biomarker discovery and explanation.
Genome Biol. 12 (6), R60.
Selhub, J., Morris, M.S., Jacques, P.F., Rosenberg, I.H., 2009. Folate-vitamin B-12 interaction
in relation to cognitive impairment, anemia, and biochemical indicators of vitamin B-
12 deficiency. Am. J. Clin. Nutr. 89 (2), 702S–706S.
Severance, E.G., Alaedini, A., Yang, S., Halling, M., Gressitt, K.L., Stallings, C.R., Origoni, A.E.,
Vaughan, C., Khushalani, S., Leweke, F.M., Dickerson, F.B., Yolken, R.H., 2012. Gastro-
intestinal inflammation and associated immune activation in schizophrenia.
Schizophr. Res. 138 (1), 48–53.
Severance, E.G., Gressitt, K.L., Stallings, C.R., Origoni, A.E., Khushalani, S., Leweke, F.M.,
Dickerson, F.B., Yolken, R.H., 2013. Discordant patterns of bacterial translocation
markers and implications for innate immune imbalances in schizophrenia. Schizophr.
Res. 148 (1–3), 130–137.
Sherwin, E., Sandhu, K.V., Dinan, T.G., Cryan, J.F., 2016. May the force be with you: the
light and dark sides of the microbiota-gut-brain axis in neuropsychiatry. CNS Drugs
30 (11), 1019–1041.
Song, X., Fan, X., Song, X., Zhang, J., Zhang, W., Li, X., Gao, J., Harrington, A., Ziedonis,
D., Lv, L., 2013. Elevated levels of adiponectin and other cytokines in drug naive,
first episode schizophrenia patients with normal weight. Schizophr. Res. 150 (1),
269–273.
Song, Y., Liu, C., Finegold, S.M., 2004. Real-time PCR quantitation of clostridia in feces of
autistic children. Appl. Environ. Microbiol. 70 (11), 6459–6465.
Sudo, N., Chida, Y., Aiba, Y., Sonoda, J., Oyama, N., Yu, X.N., Kubo, C., Koga, Y., 2004. Post-
natal microbial colonization programs the hypothalamic-pituitary-adrenal system for
stress response in mice. J. Physiol. 558 (Pt 1), 263–275.
Tan, J., McKenzie, C., Potamitis, M., Thorburn, A.N., Mackay, C.R., Macia, L., 2014. The role
of short-chain fatty acids in health and disease. Adv. Immunol. 121, 91–119.
Tomova, A., Husarova, V., Lakatosova, S., Bakos, J., Vlkova, B., Babinska, K., Ostatnikova, D.,
2015. Gastrointestinal microbiota in children with autism in Slovakia. Physiol. Behav.
138, 179–187.
Verkhratsky, A., Steardo, L., Peng, L., Parpura, V., 2016. Astroglia, glutamatergic transmis-
sion and psychiatric diseases. Adv. Neurobiol. 13, 307–326.
Wang, Y., Song, F., Zhu, J., Zhang, S., Yang, Y., Chen, T., et al., 2017 Feb. GSA: genome se-
quence archive. Genomics Proteomics Bioinformatics 15 (1):14–18. https://doi.org/
10.1016/j.gpb.2017.01.001.
Wong, J.M., de Souza, R., Kendall, C.W., Emam, A., Jenkins, D.J., 2006. Colonic health: fermen-
tation and short chain fatty acids. J. Clin. Gastroenterol. 40 (3), 235–243.
 Y. Shen et al. / Schizophrenia Research 197 (2018) 470–477 477

Yolken, R.H., Severance, E.G., Sabunciyan, S., Gressitt, K.L., Chen, O., Stallings, C., Origoni, A.,
Katsafanas, E., Schweinfurth, L.A., Savage, C.L., Banis, M., Khushalani, S., Dickerson,
F.B., 2015. Metagenomic sequencing indicates that the oropharyngeal phageome of
individuals with schizophrenia differs from that of controls. Schizophr. Bull. 41 (5),
1153–1161.
Zhang, J., Guo, Z., Xue, Z., Sun, Z., Zhang, M., Wang, L., Wang, G., Wang, F., Xu, J., Cao, H., Xu,
H., Lv, Q., Zhong, Z., Chen, Y., Qimuge, S., Menghe, B., Zheng, Y., Zhao, L., Chen, W.,
Zhang, H., 2015. A phylo-functional core of gut microbiota in healthy young Chinese
cohorts across lifestyles, geography and ethnicities. ISME J. 9 (9), 1979–1990.
Zheng, P., Zeng, B., Zhou, C., Liu, M., Fang, Z., Xu, X., Zeng, L., Chen, J., Fan, S., Du, X., Zhang,
X., Yang, D., Yang, Y., Meng, H., Li, W., Melgiri, N.D., Licinio, J., Wei, H., Xie, P., 2016. Gut
microbiome remodeling induces depressive-like behaviors through a pathway
mediated by the host's metabolism. Mol. Psychiatry 21 (6), 786–796.