Journal of Alzheimer’s Disease 63 (2018) 1337–1346 1337

DOI 10.3233/JAD-180176
IOS Press

Gut Microbiota is Altered in Patients

with Alzheimer’s Disease
Zhen-Qian Zhuanga , Lin-Lin Shena , Wei-Wei Lia , Xue Fub , Fan Zenga , Li Guic , Yang Lüb , Min Caid ,
Chi Zhua , Yin-Ling Tane , Peng Zhengf , Hui-Yun Lia , Jie Zhua , Hua-Dong Zhoua , Xian-Le Bua,∗
and Yan-Jiang Wanga,∗
a Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing, China
b Department of Geriatrics, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
c Department of Neurology, Southwest Hospital, Third Military Medical University, Chongqing, China
d Department of Neurology, Chongqing General Hospital, Chongqing, China
e Department of Microbiology, Third Military Medical University, Chongqing, China
f Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China

Handling Associate Editor: Jin-Tai Yu

Accepted 26 March 2018

Abstract. Previous studies suggest that gut microbiota is associated with neuropsychiatric disorders, such as Parkinson’s
disease, amyotrophic lateral sclerosis, and depression. However, whether the composition and diversity of gut microbiota is
altered in patients with Alzheimer’s disease (AD) remains largely unknown. In the present study, we collected fecal samples
from 43 AD patients and 43 age- and gender-matched cognitively normal controls. 16S ribosomal RNA sequencing technique
was used to analyze the microbiota composition in feces. The composition of gut microbiota was different between the two
groups. Several bacteria taxa in AD patients were different from those in controls at taxonomic levels, such as Bacteroides,
Actinobacteria, Ruminococcus, Lachnospiraceae, and Selenomonadales. Our findings suggest that gut microbiota is altered
in AD patients and may be involved in the pathogenesis of AD.

Keywords: Alzheimer’s disease, amyloid-␤ peptide, gut microbiota, 16S ribosomal RNA sequencing

INTRODUCTION dementia [3]. Although tremendous efforts have been

Alzheimer’s disease (AD) is the most common
form of dementia, causing a tremendous social and
economic burden [1, 2]. Senile plaques are thought
to be the pathological hallmark of AD, consisting of
amyloid-␤ protein (A␤), which can cause secondary
pathological changes such as hyperphosphoryla-
tion of tau, neuroinflammation, oxidative stress,
and neurite degeneration, and eventually leading to
∗ Correspondence
to: Yan-Jiang Wang and Dr. Xian-Le Bu,
Department of Neurology, Daping Hospital, Third Military Med-
ical University, Chongqing, China. E-mails: yanjiang wang@
tmmu.edu.cn; buxianle@sina.cn.
made for the treatment of AD, no efficient disease-
modifying therapeutics are available, partially due to
the limited understanding of the disease pathogene-
sis [4].
There is a large number of microbiota (most of
them are bacteria) in gut, which is ten times more
than the number of cells in human body [5], and
the total genome of gut microbiota are 150 times
more than genome of human body [6]. Amount-
ing evidence suggested that gut microbiota plays an
important role in the development of brain. And there
is a bidirectional relationship between the brain, gut,
and the microbiota within the gut, which is referred
as the microbiota-gut-brain axis [7]. The alterations
in the gut microbiota composition were proven linked

ISSN 1387-2877/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved
1338 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease
to a number of neuropsychiatric disorders including
depression, amyotrophic lateral sclerosis (ALS) and
Parkinson’s disease (PD) [7]. Specifically, gut micro-
biota can induce depression-like behavior through
altering host metabolism [8]. The proinflammatory
gut microbiota dysbiosis in PD patients could trig-
ger inflammation-induced misfolding of ␣-synuclein.
AD shared several common properties with other
neurodegenerative disorders: 1) accumulation of mis-
folded proteins (A␤ and hyperphosphorylated tau); 2)
evidence for a prion-like spread of pathology with
misfolded proteins; and 3) neuroinflammation [9].
However, whether gut microbiota participated the
pathogenesis of AD remains largely unknown. In the
present study, we collected the feces from AD patients
and cognitively normal controls and found that the gut
microbiota composition in AD patients was different
from that in normal controls.
MATERIALS AND METHODS
Subjects
Forty-three AD patients and 43 age- and gender-
matched controls with normal cognition were
recruited from Daping Hospital and Southwest Hos-
pital of Third Military Medical University, First
Affiliated Hospital of Chongqing Medical University,
and Chongqing People’s Hospital. The subjects were
excluded if they had 1) a family history of demen-
tia; 2) any kind of other neurodegenerative disease
(e.g., PD, ALS); 3) severe cardiac, pulmonary, hep-
atic, renal diseases, or any kinds of tumor; 4) enduring
mental illness (e.g., schizophrenia); 5) a history of
taking antibiotics within six months, 6) intestinal dis-
eases (e.g., irritable bowel syndrome). This study was
approved by the Institutional Review Board of Dap-
ing Hospital.
Clinical assessment and diagnosis of AD
The clinical assessment and diagnosis of AD
dementia were performed following a previously
used protocol [10, 11]. In brief, the demographic data
and medical history (such as hypertension, coronary
heart disease, diabetes mellitus and gastrointestinal
diseases) were collected. Cognitive and functional
status was assessed based on a neuropsychological
battery as described previously [12]. The cognitive
and functional status was assessed using the Chinese
version of the Mini-Mental State Examination
(MMSE) and instrumental Activities of Daily Living
(ADL). The subjects who were abnormal in MMSE
assessment were further administered with neuropsy-
chological tests, including Clinical Dementia Rating
(CDR), Fuld Object Memory Evaluation for detecting
extensive cognitive dysfunction mainly composed
of memory, Rapid Verbal Retrieve for detecting
the function of semantic memory, Wechsler Adult
Intelligence Scale (Digit Span and Block Design sub-
tests) for evaluating immediate memory and function
of graphical recognition, Pfeiffer Outpatient Dis-
ability Questionnaire for assessing ability of social
activities, Hamilton Depression Rating Scale for
measuring emotional status, and Hachinski Ischemic
Score (HIS) for evaluating significant vascular dis-
eases. The diagnosis of AD was made according
to the criteria of the National Institute of Neuro-
logical and Communicative Diseases and Stroke/AD
and Related Disorders Association. These procedures
were administered by trained interviewers who were
experienced neurologists. In addition, twelve people
were administered with A␤ positron emission tomog-
raphy (PET) inspection with Pittsburg compound B
to detect and quantify A␤ deposition in the brain.
Fecal sample collection and DNA isolation
Fecal samples were collected from the recruited
AD patients and matched controls in the morning and
stored at –80◦ C prior to analyses. DNA was extracted
using the standard Power Soil Kit according to the
product instructions [8]. The final DNA concentra-
tion and purification were determined by NanoDrop
2000 UV-vis spectrophotometer (Thermo Scientific,
Wilmington, USA), and DNA quality was checked by
1% agarose gel electrophoresis. DNA samples were
stored at –20◦ C until subsequent analyses.
16S rRNA gene sequencing and analysis
The V3-V4 region of the bacteria’s 16S ribosomal
RNA (rRNA) gene was amplified by thermocy-
cler PCR system (GeneAmp 9700, ABI, USA) with
barcode-indexed primers (338F and 806R) accord-
ing to previous protocols [14]. The PCR reactions
were conducted using the following program: 3 min
of denaturation at 95◦ C, 27 cycles of 30 s for denatu-
ration at 95◦ C, 30 s for annealing at 55◦ C, and 45 s for
elongation at 72◦ C, and a final extension at 72◦ C for
10 min. PCR reactions were performed in triplicate
20 ␮L mixture containing 4 ␮L of 5 × FastPfu Buffer,
2 ␮L of 2.5 mM dNTPs, 0.8 ␮L of each primer
(5 ␮M), 0.4 ␮L of FastPfu Polymerase and 10 ng
 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease 1339

Table 1
Characteristics of study population
Characteristics AD patients (n = 43) Normal controls (n = 43) p
Age, y (SD) 70.12 (8.78) 69.72 (9.24) 0.839
Female, n (%) 20 (46.5) 20 (46.5) 1
Education, y (SD) 9.23 (2.15) 9.81 (2.52) 0.253
Hypertension, n (%) 15 (34.9) 13 (30.2) 0.645
Diabetes mellitus, n (%) 7 (16.3) 5 (11.6) 0.534
Hypercholesterolemia, n (%) 11 (25.5) 10 (23.3) 0.802
Coronary heart disease, n (%) 8 (18.6) 6 (14) 0.559
MMSE scores, mean (SD) 14.70 (4.94) 27.78 (2.54) <0.001
CDR score, mean (SD) 1.67 (0.84) 0.00 (0.00) <0.001
ADL score, mean (SD) 48.63 (18.03) 21.18 (3.17) <0.001
APOE ␧4 carriers, n (%) 17 (39.5) 10 (23.3) 0.009
AD, Alzheimer’s disease; BMI, Body mass index; MMSE, Mini-Mental State Examination; CDR, Clinical
Dementia Rating; ADL, Activities of Daily Living; All data, except for gender, Hypertension, Diabetes melli-
tus, Hypercholesterolemia, Coronary heart disease and APOE genotype, are expressed as means (SD). pvalue,
two-dependent t test or Chi-squared test were as appropriate.

of template DNA. The resulted PCR products were
extracted from a 2% agarose gel and further purified
using the AxyPrep DNA Gel Extraction Kit (Axygen
Biosciences, Union City, CA, USA) and quantified
using QuantiFluor™-ST (Promega, USA) according
to the manufacturer’s protocol. Purified amplicons
were pooled in equimolar and paired-end sequenced
(2 × 300) on an Illumina MiSeq platform (Illumina,
San Diego, USA) according to the standard protocols
by Majorbio Bio-Pharm Technology Co. Ltd. (Shang-
hai, China). The raw reads were deposited into the
NCBI Sequence Read Archive (SRA) database.
Sequence analysis
The final 86 samples from AD patients and healthy
group were pooled into four libraries according to 16S
rRNA sequencing data. The 3273141 qualified reads
from 6546282 raw reads were filtered for downstream
analysis. After filtering, an average of 38060 reads
per sample was obtained (minimum 30460; maxi-
mum 44928). Then all remaining reads were assigned
to operational taxonomic units (OTUs) with a 97%
threshold of distance-based similarity and classified
according to the RDP reference database. Summaries
of the taxonomic distributions of OTUs were con-
structed with these taxonomies and were used to
calculate the relative abundances of microbiota at
different taxonomic levels.
Statistical analysis
Alpha diversity, including the Shannon Index,
observed species, Simpson and Chao1 indices,
were investigated in QIIME, and the significance
was determined using a t test. Distance matrices
(Beta diversity) were generated based on weighted
and non-weighted algorithms and generated prin-
cipal coordinate analysis (PCoA) in R software
(Version 2.15.3). PLS-DA was performed using
SIMCA-P software to cluster the sample plots
across groups. To perform the UniFrac analysis,
representative sequences for each OTU were aligned
using PyNAST and a phylogenetic tree from this
alignment was constructed with Fast Tree. Random
Forest algorithm was carried out to identify the key
discriminatory OTUs which assigns an importance
score to each OTU by estimating the increase in
error caused by removing that OTU from the set
of predictors. Linear discriminant analysis with
effect size (LEfSe) and phylogenetic investigation
of communities by reconstruction of unobserved
states (PICRUSt) were performed online (http://
huttenhower.sph.harvard.edu/galaxy/), based on
the taxonomic files obtained from the QIIME
analysis.
RESULTS
Subject characteristics
The characteristics of AD patients and age and
gender-matched cognitively normal controls are
shown in Table 1. AD patients had higher frequency
of APOE ␧4 carriers, lower MMSE score, and higher
CDR and ADL score. No significant difference was
found in education and comorbidities of hyperten-
sion, diabetes mellitus, hypercholesterolemia, and
coronary heart disease between AD patients and nor-
mal controls.
1340 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease
Fig. 1. Beta diversity measures between AD and control group. a–d: PLS-DA analysis at the family (a), genus (b), species (c) and OTU (d)
level.
Bacterial community structure in AD and control
group
To examine the bacterial community structure in
the stool samples, the dataset were analyzed by Par-
tial Least Squares Discriminant Analysis (PLSDA).
Fecal microbial communities separated between AD
and control group on family, genus, species and OTU
level, suggesting a unique gut microbiota in AD
(Fig. 1).
The subgroup analysis including AD patient who
underwent PET and cognitively normal controls, also
showed an obvious unique gut microbiota in AD with
PLSDA. In this subgroup analysis, twelve age- and
gender-matched cognitively normal subjects were
selected as controls. These results were essentially
the same as that from the whole subjects (Fig. 2).
Composition of gut microbiota in AD and control
groups
The overall microbial compositions of AD and
control groups were examined at different taxonomic
levels. At the phylum level, Firmicutes, Proteobac-
teria, Bacteroidetes and Actinobacteria were the
dominant bacteria (Fig. 3a). A mild decrease was
observed in the abundance of Bacteroidetes among
AD patients (p = 0.039), whereas Actinobacteria
were slightly more abundant. While Verrucomicrobia
decreased in AD patients, yet it is not statistically sig-
nificant (p = 0.15). Firmicutes were almost the same
between the two groups. At the class level, Clostridia,
Gammaproteobacteria, Bacteroidia, Bacilli, Neg-
ativicutes and Actinobacteria were the dominant
bacteria (Fig. 3b). We found that the relative abun-
dance of Actinobacteria and Bacilli increased, while
that of Negativicutes and Bacteroidia decreased
significantly in the AD group compared with the
control group. At the order level, Clostridiales,
Enterobacteriales, Bacteroidales, Lactobacillales,
and Selenomonadales were the dominant bacteria
(Fig. 3c). The relative abundance of Lactobacillales
increased, while Bacteroidales and Selenomonadales
decreased in the AD group compared with the control
group.
At the family level, Lachnospiraceae, Ruminococ-
caceae, Enterobacteriaceae, Bacteroidaceae, Veil-
lonellaceae, Erysipelotrichaceae, and Enterococ-
caceae were the dominant bacteria (Fig. 3d). In AD
patients, the relative abundance of Ruminococcaceae,
 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease 1341

Fig. 2. Beta diversity measures between PET-AD and control group. a–d: PLS-DA analysis at the family (a), genus (b), species (c) and OTU
(d) level.

Fig. 3. Composition of the gut microbiota in AD and control group. Relative abundance of phylum-level (a), class-level (b), order-level
(c) and family-level (d) gut microbial taxa.
1342 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease

Fig. 4. Differences of bacterial taxa between AD and control group. a) Taxonomic represents statistically differences between AD and
control group. Differences are represented by the color of the most abundant class. The diameter of each circle’s diameter is proportional to
the taxon’s abundance. b) Histogram of the LDA scores for different abundant genera. Positive LDA scores indicate the enrichment of taxa
in AD group (red) relative to control group (green), and negative LDA scores indicate the depletion of taxa in AD group relative to control
group. Box shows statistically significant different bacteria. On the phylum level (c), on the class level (d), on the genus level-selected major
taxa (e), on the species-selected major taxa (f). Each box represents the 25th to 75th percentiles. (Pairwise comparisons using Wilcoxon rank
sum test; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001).
 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease
Enterococcaceae, and Lactobacillaceae increased,
while that of Lanchnospiraceae, Bacteroidaceae,
and Veillonellaceae decreased significantly com-
pared with the control group.
The microbial composition for PET subgroup was
shown in Supplementary Figure 1 in the supplemental
material. They were essentially the same with that of
the overall subjects.
Crucial bacteria associated with AD
A representative cladogram of fecal microbial
structure indicated a significant gut microbial imbal-
ance in AD. We used LEfSe to identify the taxa
that were significantly different between groups. In
our study, taxa with an average relative abundance
over 0.0001 were selected for LEfSe. This threshold
ensured that meaningful taxa were retained and the
rarest taxa were removed. The characteristics of these
comparisons are presented in Fig. 4. The LEfSe for
PET subgroup were shown in Supplementary Fig-
ure 2 in the supplemental material.
We further analyzed the variant taxa that occupy
high abundance at different taxonomic levels. At the
phylum level, we found differences between groups
in Bacteroidetes and Actinobacteria. The subspecies
of Bacteroidetes in other taxonomic levels were also
different. Although Firmicutes were almost the same
between the two groups, there still was a difference
in Firmicutes/Bacteroidetes ratio, which was reported
increased in subjects with obesity [15].
At the class level, the results were almost the same
as phylum level, but we found a significant decrease
of Negativicutes in AD group in comparison to nor-
mal controls.
At the family level, while Lachnospiraceae
was decreased in AD group, there was an
increase in Ruminococcaceae. Lachnospiraceae, and
Ruminococcaceae are two high abundance taxa
in Clostridiales, and are two important digestive
bacteria in human body. Additionally, the change
of Lachnoclostridium and Subdoligranulum, which
belonged to their subspecies respectively at the genus
level, showed the similar results.
DISCUSSION
In the present study, the gut microbiota composi-
tion and diversity were found altered in AD patients
compared with cognitively normal controls. Several
bacterial taxa, such as Actinobacteria, Bacteroidales,
1343
Ruminococcaceae, Selenomonadales, and Lachno-
clostridium, may contribute to the differences.
The balance of bacteria in the gut plays an
important role in human health. Increasing evidence
showed that gut microbes can influence brain function
and behavior via the microbiota-gut-brain axis [16].
Using germ-free animals, antibiotics or probiotics
intervention and fecal microbiota transplantation sug-
gested a role for the gut microbiota in the regulation
of mood, anxiety, pain and cognition [17]. The gut
microbiota may communicate with brain through
neural, endocrine and immune pathways [17, 18].
A number of neuropsychiatric disorders including
depression, ALS, and PD have been proved to be
related to gut microbiota [7]. Moreover, gut micro-
biota was reported to modulate brain inflammatory
pathways via the inflammasome signaling [19]. Some
probiotics in gut could modulate the expression of
specific genes in the brain [20]. Gut microbiota could
also influence neural development [16]. Moreover,
recent study indicated that microbial products can be
translocated into the brain and modulate brain gene
expression [21].
Emerging evidence suggested a link between gut
microbiota, cognition, and AD. Previous animal stud-
ies show that Bifidobacteria has a positive impact on
cognition of an anxious mouse strain after daily feed
for 11 weeks [22], and the composition and diversity
of gut microbiota were changed in AD transgenic
mice compared with wild-type mice [23]. A recent
study showed that gut microbiota was altered in AD
patients [24]. The present study further provided the
human evidence that the gut microbiota composition
was altered in AD patients, especially in PiB-PET
positive AD patients. It is worthy of noting that the
detailed changes of the gut microbiota of AD patients
in this study is different from ours. This discrep-
ancy might be due to the sample size, population,
lifestyle, dietary habits, RNA sequencing area, and
comorbidity burden [25], which is associated with gut
microbiota, between the two studies. However, the
underlying mechanisms remained poorly understood.
The gut microbiota emerged as an important reg-
ulator of host immune function and metabolism.
Changes in the composition of the gut microbiota
could increase intestinal permeability and induce
inflammation that has been proved to increase the
risk of AD [17]. In this study, we found a lower
Bacteroides abundance in AD patients, which is
consistent with a previous study showing that Bac-
teroides fragilis was lower in patients with cognitive
impairment and brain amyloidosis [26]. However, the
1344 Z.-Q. Zhuang et al. / Gut Microbiota and Alzheimer’s Disease
Bacteroides fragilis was not detected in this study,
partially due to its low abundance which it is esti-
mated as only 0.5% of the Bacteroides species in the
gut and make it technically difficult to be detected
[27]. Bacteroides fragilis can be classified into two
strains: non-toxigenic Bacteroides fragilis (NTBF)
and enterotoxigenic Bacteroides fragilis (ETBF).
ETBF, a kind of anti-inflammatory bacteria, has the
ability to strengthen intestinal barrier and reverse
gut leakiness [28]. On the other hand, Ruminococ-
cus species (increased in AD patients in the present
study), a Gram-positive bacteria, need fermentable
carbohydrates for growth, and can degrades mucus
by expressing intramolecular trans-sialidases [29]. It
is worthy of noting that bacterial components were
found in the brain of AD patients [30, 31]. It is
intriguing to speculate that gut leakiness due to gut
microbiota alteration may facilitate the entrance of
gut bacteria and their components into the body.
It has been proved that some metabolites of
gut microbiota can induce neuroinflammation [32].
Recent study showed that changing the gut microbial
composition and diversity of APP/PS1 mice by long-
term broad-spectrum antibiotics could decrease brain
A␤ burden via regulating host innate immunity [33,
34]. We previously found that AD is associated with
infectious burden [12]. Infection with pathogenic
microbes like Helicobacter pylori may exacerbate
brain A␤ production and tau phosphorylation and
increase the risk of AD [35, 36].
Gut microbiota play important roles in the synthe-
sis of brain neurotransmitters such as ␥-aminobutyric
acid (GABA), serotonin, and brain-derived neu-
rotrophic factor which are all related with AD [37].
Some gut microbes can also produce metabolites such
as neurotransmitters, neuropeptides, endocrine hor-
mones, and immunomodulators [38]. Ruminococcus
in gut, which was increased in AD patients of the
present study, was suggested to predict lower brain
N-acetylaspartate, a biochemical indicator of neu-
ronal health and is decreased in neuron metabolic
disturbance [39]. The increase of Ruminococcus
species in AD patients provided a proof of interac-
tion between gut microbiota and brain metabolites.
Metabolic diseases including obesity, insulin resis-
tance, hypertension, and hyperlipidemia are related
to AD development [40]. Meanwhile, accumulating
evidence indicated a link between gut microbiota
and metabolic disease [38]. So the alternations of
gut microbiota might contribute to AD pathogene-
sis through metabolic pathways. Additionally, a large
amount of bacteria in human intestine could gen-
erate amyloid peptides [41, 42]. As to our finding
that peripheral A␤ can enter brain and cause AD-
type pathologies [43], gut microbiota might directly
contribute to brain A␤ deposition with bacterial
amyloids.
Food style and comorbidities such as hypertension,
hyperlipidemia, and diabetes mellitus may influence
the composition of gut microbiota. In the present
study, all AD patients and control subjects were
recruited from same area and shared similar food
habit. In addition, there were no difference on the inci-
dence of hypertension, hyperlipidemia, and diabetes
mellitus between AD patients and controls. Thus, the
alteration of gut microbiota in AD patients may not be
resulted from the differences in food style and comor-
bidities. A major limitation of the present study is that
our study is a cross-sectional study. We cannot draw
conclusion about the causal relationship between the
alteration of gut microbiota and AD. Longitudinal
studies are needed to better clarify the impact of gut
microbiota on AD in the future.
In summary, our study revealed the alternation of
gut microbiota composition in AD patients. Modu-
lation of gut microbiota through personalized diet or
beneficial microbiota intervention may be a poten-
tial strategy for the prevention treatment of AD. Our
study also suggests that AD might not be a disease of
brain itself, and brain health is closely associated with
our whole body. We need to understand the disease
pathogenesis and develop therapies systemically for
AD and other neurodegenerative diseases [44, 45].
ACKNOWLEDGMENTS
This study was supported by National Natural
Science Foundation of China (NSFC) Grant No.
91749206, 81701043 and 81625007.
Authors’ disclosures available online (https://
www.j-alz.com/manuscript-disclosures/18-0176r1).
SUPPLEMENTARY MATERIAL
The supplementary material is available in the
electronic version of this article: http://dx.doi.
org/10.3233/JAD-180176.
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