Journal of Alzheimer’s Disease xx (20xx) x–xx 1

DOI 10.3233/JAD-190587
IOS Press

1 Analysis of Salivary Microbiome in Patients

2 with Alzheimer’s Disease

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3 Xi-Xi Liua,b,1 , Bin Jiaoa,b,c,1 , Xin-Xin Liaob,d , Li-Na Guoa,b , Zhen-Hua Yuana,b , Xin Wanga,b ,
4 Xue-Wen Xiaoa,b , Xin-Yue Zhanga,b , Bei-Sha Tanga,b,c,e,f,g and Lu Shena,b,c,h,∗
5
a Department of Neurology, Xiangya Hospital, Central South University, Changsha, China
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b National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University,
7 Changsha, China

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c Key Laboratory of Hunan Province in Neurodegenerative Disorders, Central South University, Changsha, China

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d Department of Geriatrics Neurology, Xiangya Hospital, Central South University, Changsha, China

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e Parkinson’s Disease Center of Beijing Institute for Brain Disorders, Beijing, China

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f Collaborative Innovation Center for Brain Science, Shanghai, China
g Collaborative Innovation Center for Genetics and Development, Shanghai, China

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h Key Laboratory of Organ Injury, Aging and Regenerative Medicine of Hunan Province, Changsha, China

Accepted 11 September 2019

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14 Abstract. Recent studies found that poor oral hygiene was associated with increased risk of dementia, and the number
15 of oral bacteria significantly increased in the brain tissues of patients with Alzheimer’s disease (AD), suggesting that the
16 oral microbiota may play an important role in the pathogenesis of AD. However, the actual composition of oral bacteria
17 communities in patients with AD and whether these oral bacteria are associated with disease severity remain largely unknown.
Also, the APOE ␧4 polymorphism is a strong risk factor for sporadic AD, and it would be pertinent to see if the bacterial flora
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19 was different in those patients who were APOE ␧4 positive. A total of 78 subjects were recruited in this study, including 39
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20 patients with AD and 39 healthy controls. Saliva was collected from each subject. 16S ribosomal RNA (16S rRNA) sequencing
21 was conducted to analyze the salivary microbiota, and Sanger sequencing was performed to analyze the APOE genotype.
22 There was a significantly lower richness and diversity of saliva microbiota detected in AD patients than healthy controls. The
23 relative abundance of Moraxella, Leptotrichia, and Sphaerochaeta in the saliva of AD patients greatly increased, whereas that
24 of Rothia was significantly reduced. Compared with APOE ␧4 (–) patients, the level of Abiotrophia and Desulfomicrobium
was comparatively abundant, while Actinobacillus and Actinomyces decreased significantly in patients carrying the APOE ␧4.
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26 No bacteria were found to be associated with the severity of AD. This is the first study to analyze the salivary microorganisms
27 in patients with AD, and we discovered that the composition of salivary microbiome was altered in AD, providing further
28 support for the role of the oral microbiome in AD development.
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29 Keywords: Alzheimer’s disease, APOE, oral microbiome, 16S rRNA

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30 INTRODUCTION tia worldwide. Alzheimer’s disease (AD) is the 33

most common form of dementia [1]. It is a neu- 34

31 According to the 2018 World Alzheimer Report, rodegenerative disease characterized by progressive 35

32 there are 50 million people living with demen- cognitive dysfunction and abnormal mental behavior 36

[2]. The two diagnostic neuropathological hallmarks 37

1 These are numerous extracellular deposits of amyloid-␤ 38

authors contributed equally to this work.
∗ Correspondence to: Dr. Lu Shen, Department of Neurology, (A␤) plaques and neurofibrillary tangles [3]. It is 39

Xiangya Hospital, Central South University, Changsha, China. believed that AD is a multifactorial disease primarily 40

E-mail: shenlu@csu.edu.cn. or E-mail: shenlu2505@126.com. caused by genetic, aging, and environmental factors 41

ISSN 1387-2877/19/$35.00 © 2019 – IOS Press and the authors. All rights reserved
 2 X.-X. Liu et al. / Salivary Microbiome in AD

42 [4–6]. There is no cure for AD yet, partially due to age and gender. The written informed consent for 91

43 the reason that the pathogenesis of AD is still unclear. participation in the study was obtained from all 92

44 Accumulating evidence has demonstrated that subjects. This study was conducted in accordance 93

45 tooth loss and poor oral hygiene are important risk with the Declaration of Helsinki and was approved 94

46 factors for dementia [7, 8]. Previous studies have by the Ethical Review Board at Xiangya Hospital of 95

47 shown that periodontal disease is associated with Central South University in China. 96

48 AD [9, 10]. More recently, Taiwanese studies have

49 found a strong link between chronic periodontitis and Clinical data 97

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50 risk of AD [11, 12]. Evidence of oral bacteria was

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51 also found in the brain tissue and cerebrospinal fluid Neuropsychological assessment was performed on 98

52 of autopsy cases with histopathologically-confirmed all patients, including Mini-Mental State Examina- 99

53 AD [13–15] and oral bacteria were present at about tion (MMSE), Neuropsychiatric Inventory Question- 100

54 7-fold higher density and far greater variety in AD naire (NPI), Clinical Dementia Rating Scale (CDR), 101

55 brains compared to cognitively normal controls [16]. and Activity of Daily Living Scale (ADL). MMSE 102

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56 These findings suggest that the disorder of oral micro- was also conducted with healthy controls that met 103

57 biome may increase the infection of opportunistic the inclusion criteria. 104

58 pathogens in the brain of AD patients and thus con-

59 tributes to the development of AD. However, the APOE genotyping 105

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60 actual composition of oral bacteria communities in
61 patients with AD remains unknown. In this study, we Blood samples were collected from all patients 106

62 first conducted the analysis of salivary microbiome in using ethylenediaminetetraacetic acid (EDTA) tubes. 107

63 AD and further investigated whether it is influenced Genomic DNA was extracted using the standard 108

64 by APOE genotypes and disease severity by using phenol-chloroform extraction method. All DNA sam- 109
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65 16S ribosomal RNA (16S rRNA) gene sequencing. ples were diluted to 50 ng/␮l. A 581-bp fragment 110

was amplified using the following primers: forward 111

66 MATERIALS AND METHODS 5 - CCTACAAATCGGAACTGG -3 , and reverse 112

5 - CTCGAACCAGCTCTTGAG -3 . Polymerase 113

67 Participants chain reaction (PCR) was performed as previously 114

described [18]. Each PCR product was sequenced on

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68 This study recruited a total of 39 AD patients and an ABI 3730xl DNA analyzer (ABI, USA). 116
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69 39 age-matched healthy controls. The diagnoses of

70 probable or possible AD according to the NINCDS- Saliva sample collection and DNA isolation 117

71 ADRDA criteria [17] were made by two or more

72 experienced neurologists in Xiangya Hospital. The Approximately 2 ml spontaneous, whole unstim- 118

73 subjects were excluded if they had: other causes of ulated saliva was collected from each subject and 119
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74 dementia or other types of dementia; any kind of stored at –80◦ C prior to analyses. All the subjects 120

75 other neurodegenerative diseases (e.g., Parkinson’s were banned from consuming food and drink for 121

76 disease); severe cardiac, pulmonary, hepatic, renal more than 2 h before sampling. Saliva DNA was 122

77 diseases, any kinds of tumor, rheumatoid immune- extracted using QIAamp DNA Investigator Kit (Qia- 123
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78 related diseases (e.g., Sjogren’s syndrome); a history gen, USA) according to the product instructions. 124

79 of smoking, alcohol drinking, taking antibiotics, The nucleic acid quantitative analysis was per- 125

80 glucocorticoids, or probiotics within one month. We formed using Qubit dsDNA HS Assay Kit (Thermo 126

81 also had an oral specialist help us to exclude subjects Scientific Inc, USA). 127
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82 with the following conditions within two months:

83 oral surgery or dental procedure, inflammation of 16S rRNA gene sequencing and analysis 128

84 oral or perioral tissues, and other chronic disease

85 in the oral cavity. Periodontal diseases were not S rRNA high-variable regions V3-V4 were 129

86 excluded and we did not systematically evaluate amplified using thermocycler PCR system (Veriti 130

87 the oral or dental condition of the participants. ABI, USA) with primers (Pro341F and Pro805R) 131

88 In order to minimize bias due to different dietary according to the previous protocols [19], and then 132

89 structure, the control group was all from the spouses sequenced with Illumina Hiseq2500. The raw 133

90 or caretakers living with AD patients matched with sequencing reads were detected by FastQC software 134
 X.-X. Liu et al. / Salivary Microbiome in AD 3

Table 1 Alpha diversity analysis 170

Demographic information of AD patients and control groups∗
Subjects Age Male/Female MMSE A total of 5,857,141 high-quality sequences were 171

Total 64.41 ± 10.04 18/21 14.31 ± 7.14 obtained for OTU clustering based on the refer- 172
APOE ␧4(+) 65.00 ± 10.13 12/11 13.40 ± 8.54 ence database. Among them, 5,655,352 (96.55%) 173
APOE ␧4(–) 63.36 ± 10.07 6/10 16.33 ± 6.34
AD Patients
sequences were matched to 97% sequence similar- 174

Mild AD 65.93 ± 9.6 6/7 23.00 ± 2.09 ity, with a total of 16,077 OTUs. Good’s estimator of 175

Moderate AD 67.25 ± 9.32 8/4 15.08 ± 2.87 coverage was 99.17%, indicating that the 16S rRNA 176

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Severe AD 59.67 ± 8.91 4/10 5.40 ± 1.84 sequences identified in this study likely represent the 177
63.90 ± 9.36 28.38 ± 1.21

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Healthy controls 21/18
∗ Data
majority of bacterial sequences present in the saliva 178
are reported as mean ± SD.
samples. Alpha diversity analysis showed that there 179

was a significantly lower richness (Chao1 index) and 180

diversity (Shannon index, PD whole tree index) of 181

135 to remove the primer region and low-quality saliva flora detected in AD patients than in healthy 182

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136 sequences. The chimera sequences arising from controls (Fig. 1). 183

137 the PCR amplification were detected and excluded

138 using Mothur (http://www.mothur.org) based on the Community composition 184

139 GreenGenes database. The high-quality reads that

AD and control group

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140 reached a 97% nucleotide similarity were clustered 185

141 into operational taxonomic units (OTUs) according The salivary bacteria community composition of 186

142 to the Ribosomal Database Project (RDP) database. AD patients and healthy controls were analyzed at 187

143 Alpha diversity analyses, including Rarefaction different taxonomic levels. The overall microbial 188

144 curve, Shannon index, observed species, Chao1, and community composition of two groups at the phy- 189
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145 Phylogenetic Diversity whole tree (PD whole tree) lum level was similar (Fig. 2A). At the genus level, 190

146 indices, were investigated in QIIME, and the sig- among most of the predominant genera, the rela- 191

147 nificance was determined using t-test. Composition tive abundance of Moraxella increased significantly 192

148 and beta diversity analyses were then conducted in AD patients (p < 0.05). The genera with aver- 193

149 to compare the differences of salivary microbiome age relative abundance >0.1% were demonstrated in 194

Fig. 2B, C.
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150 between groups. Tree analysis and principal coordi- 195

151 nate analysis (PCoA) were conducted based on the

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152 weighted UniFrac algorithm (http://unifrac.colorado. AD patients with different APOE genotype and 196

153 edu/). Linear discriminant analysis with effect size severity 197

154 (LEfSe) and Metastats analysis were performed The subgroup analyses showed that among the pre- 198

155 online (http://huttenhower.sph.harvard.edu/lefse/ dominant genera, Actinobacillus and Actinomyces in 199

156 and http://metastats.cbcb.umd.edu/, respectively), the saliva of patients carrying the APOE ␧4 decreased 200
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157 based on the taxonomic files obtained from the significantly (p < 0.05) (Fig. 3A, B). However, no bac- 201

158 QIIME analysis. teria were found to be associated with the severity of 202

AD. 203
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159 RESULTS Beta diversity analysis 204

160 Demographic information To examine the bacterial community structure in 205

all saliva samples, a beta diversity analysis indicating 206

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161 The demographic information of AD patients and the differences among groups was conducted. 207

162 healthy controls are shown in Table 1. In order to

163 compare the differences in oral bacteria between AD and control group 208
164 the patients with different severity and with differ- Due to interindividual variation, the saliva micro- 209
165 ent APOE genotypes, all the patients were divided biotas of the two groups could not be divided into 210
166 into three groups according to the MMSE score clusters using weighted UniFrac metrics and could 211
167 (Mild: MMSE ≥20, Moderate: 10≤ MMSE <20, not be separated clearly by PCOA (see Supplemen- 212
168 and Severe: MMSE <10) and were also divided into tary Figures 1 and 2). The metagenome analysis 213
169 APOE ␧4 (+) group and APOE ␧4 (–) group. LEfSe approach was further applied to identify the 214
4 X.-X. Liu et al. / Salivary Microbiome in AD

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Fig. 1. Alpha diversity analysis for AD patients (P) and healthy controls (C). Compared with the healthy controls, the richness and diversity
of saliva flora in AD patients were significantly reduced (p < 0.05). A) Rarefaction curves, B-D) Boxplots of observed species/OTUs, Chao1
index, Shannon index, PD whole tree (∗∗∗ p < 0.001).

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Fig. 2. Relative abundance of bacterial phyla and predominant genera (>0.1%) among AD patients (P) and healthy controls (C). A) Phylum
level. The community composition of the two groups at the phylum level was similar. B) Comparison of the top 8 abundant genera. C)
Comparison of the top 9 to 30 abundant genera. Only the relative abundance of Moraxella increased significantly in AD patients (∗ p < 0.05).
 X.-X. Liu et al. / Salivary Microbiome in AD 5

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Fig. 3. Metastats analysis of taxa in AD patients with different APOE genotype (APOE ␧4 (–) and APOE ␧4 (+)). A) Family level. B) Genus
level. (∗ p < 0.05).

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215 key taxa responsible for the difference between animal models and in patients [23–25]. As one of the 250

216 groups. Moraxella, Leptotrichia, and Sphaerochaeta, most relevant microbial habitats, the oral cavity is 251

217 which were abundant in the AD patients, and Rothia colonized by a personalized set of microorganisms 252

218 which were abundant in the healthy controls, were including bacteria [26]. Besides oral disease, oral 253

the key bacteria that contributed to the difference bacteria have been proven to present a significant risk

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219 254

220 in the salivary microbiota composition between AD factor for human health, such as tumor, diabetes mel- 255

221 patients and the healthy controls (Fig. 4A, B). litus, and cardiovascular diseases [27–29]. Compared 256

with the gastrointestinal tract, the oronasal cavity 257

222 AD patients with different APOE genotype and has a close direct association with the brain via the 258
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trigeminal and olfactory nerves [14, 30]. Neverthe-
223 severity 259

224 In addition to this, we also used LEfSe to analyze less, only a few studies focused on the relationship 260

225 the subgroup and the results are shown in Fig. 4C and between the oral microbiome and neurodegenerative 261

226 D. In APOE ␧4 (+) patients, Abiotrophia and Desul- disorders such as PD [31], and the structure of oral 262

227 fomicrobium were abundant, and yet Actinobacillus microbiome in AD remains unknown. 263

showed Actinobacillus and Actinomyces decreased In the present study, the alpha analysis showed that
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228 264

229 significantly in patients carrying the APOE ␧4. Again, the diversity of salivary microbiome in AD patients 265

no taxa were found to have a significant contribu- was significantly lower compared with the healthy
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230 266

231 tion to the difference between patients with different controls. To date, this is the first study to analyze 267

232 severity according to LEfSe. the salivary microbiome in AD patients. However, 268

some studies have examined the gut microbiome in 269

233 DISCUSSION AD patients. Nicholas et al. compared the gut micro- 270
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biomes of AD patients and healthy controls using 16s 271

234 Only about 10% of cells in our bodies are from rRNA sequencing, and found that the bacteria diver- 272

235 the human host, and the rest are from human micro- sity of AD patients was significantly reduced [24], 273

236 biota. These symbiotic microorganisms help us resist which is consistent with our results obtained in saliva. 274

Despite the fact that the composition and structure of

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237 pathogens, educate the immune system, and synthe- 275

238 size nutrients, which perform important functions in oral and gastrointestinal microbiota are different, the 276

239 the human body [20]. Recently, emerging evidence bacteria from these locations may have interaction 277

240 suggested a link between human microbiota and since the alimentary tract is a continuous tube run- 278
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241 neurodegenerative diseases. Sampson et al. first con- ning from the oronasal cavity to the anus. Liang et 279

242 firmed that the gut bacteria could regulate movement al. also analyzed the fecal microbiota composition 280

243 disorders in a mouse model of PD [21]. Subsequently, in APP/PS1 transgenic mice and wild-type mice and 281

244 Ewen et al. discovered that the gut microbes of young found that the microbiota diversity of APP/PS1 mice 282

245 killifish could extend the lifespans of older fish [22], decreased with increased age [23]. On the contrary, 283

246 which give us a clue that human microbiota may also Emery et al. assessed the brain tissue from 8 post- 284

247 be involved in age-related diseases, for example, AD. mortem AD patients and 6 normal controls and found 285

248 As a result, subsequent studies have found evidence an increase in the bacterial populations in AD brain 286

249 that the gut microbes are associated with AD both in tissue compared with the normal [32]. This provides 287
 6 X.-X. Liu et al. / Salivary Microbiome in AD

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Fig. 4. LEfSe results on saliva microbiomes in different groups. A) Histogram of the LDA scores between AD patients (P) and healthy
controls (C). AD-enriched taxa are indicated with a positive LDA score (green), and taxa enriched in healthy controls have a negative score
(red). B) Taxonomic cladogram obtained from LEfSe analysis. The cladogram reports the taxa (highlighted by small circles and by shading;
red indicating healthy controls, green indicating AD patients, yellow indicating non-significant) showing different abundance values. C, D)
d

Analysis of APOE ␧4 (–) and APOE ␧4 (+) groups.

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288 further evidence of possible bacterial infection in AD with A␤ plaques [38, 39]. The taxa that signifi- 309

289 brain and the most likely source of these bacteria is cantly increased in AD patients in our study are either 310

290 the migration of bacteria in other parts of our bodies Gram-negative bacteria or bacteria with prokaryotic 311

291 [33]. functional amyloids, once they pass through the BBB 312
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292 Both the Metastats and LEfSe analysis showed or along the nerve into the brain, they may increase 313

293 that the community composition of AD patients was the deposition of A␤ plaques. 314

294 altered compared with the healthy controls. Taxa like APOE ␧4 is the main genetic risk factors for spo- 315

295 Moraxella, Leptotrichia, Sphaerochaeta, and Rothia radic AD [40]. Recently, a research demonstrated that 316

might be the key bacteria that contribute to the differ- APOE ␧4 could activate matrix metalloproteinase
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296 317

297 ence. Among these bacteria, Moraxella is the most 9 (MMP9) via cyclophilin A (CypA) [41]. MMP9 318

298 prominent. Due to the gradual decline of humoral is an enzyme that can lead to BBB breakdown. 319

299 and cell-mediated immune responses with age, our Therefore, the brain tissue of APOE ␧4 carrier is 320
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300 bacterial load steadily increases [34]. Many bacteria more vulnerable to bacterial invasion. We also found 321

301 have fimbriae, curli, and other cellular appendages the differences in salivary microbiome composition 322

302 called prokaryotic functional amyloids, can act as between APOE ␧4 (–) and APOE ␧4 (+) patients. 323

303 oligomeric toxins [35]. There is a potential for these Abiotrophia is reported to be a cause of infective 324

304 prokaryotic A␤-like fibers to cross the blood-brain endocarditis according to the Human Microbiome 325

305 barrier (BBB) and form pathological senile plaques Project [42]. Desulfomicrobium belongs to obligate 326

306 seen in AD [36, 37]. Furthermore, lipopolysaccharide anaerobe and it is reported that obligate anaerobe 327

307 from the outer membrane of Gram-negative bacte- is more common in the brain tissue of AD patients 328

308 ria was detected in AD brains and was co-located [16]. Unfortunately, no taxa were found to be 329
 X.-X. Liu et al. / Salivary Microbiome in AD 7

330 associated with disease severity, this may be due to to L.S., No.81701134 to B.J.), the National Key 379

331 the reason that the alternations in saliva microbiota R&D Program of China (No.2017YFC0840100 and 380

332 are independent of the severity of AD. Further study No.2017YFC0840104 to L.S.), the Xiangya Hospi- 381

333 should be conducted to provide more solid evidence. tal Youth Scientific Research Fund (No.2016Q01 to 382

334 None of the bacteria found in this study have been B.J.). 383

335 reported to elevate in the brains of AD patients yet. Authors’ disclosures available online (https:// 384

336 This may be due to the limitations of current detec- www.j-alz.com/manuscript-disclosures/19-0587r1). 385

337 tion methods, specific detections of these bacteria

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338 in the brain are needed in future research. On the

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339 other hand, the main dominant flora has not been
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