Despite the proposed role of gut microbiota in the development or progression of AD4,5,23, there have been no comprehensive surveys of the gut microbiome in individuals with AD. In this study, we performed bacterial 16S rRNA gene sequencing on DNA isolated from fecal samples in order to compare the composition of the gut microbiome in participants with and without a diagnosis of dementia due to AD. We discovered that the gut microbiome of AD participants has decreased microbial richness and diversity and a distinct composition compared to asymptomatic age- and sex-matched Control participants. We also identified several broad taxonomic differences between AD and Control groups, and determined that levels of differentially abundant genera correlate with CSF biomarkers of AD pathology.

The decreased richness and diversity in our study broadly parallels results observed in other conditions linked to gut microbiome alterations, including obesity, diabetes, IBD, and Parkinson’s disease13,24,25,26. Furthermore, PICRUst analysis revealed broad functional changes in predicted metabolism, bacterial cell motility, and signal transduction pathways in the gut microbiome of AD participants. While the specific bacteria responsible for these compositional and functional alterations may differ between conditions, it has been proposed that these broad-scale changes in gut microbiota (often referred to as “dysbiosis”) may play important roles in disease progression and maintenance, potentially through immune activation and systemic inflammation27. While it is unclear how the gut influences the development of neuropathology, substantial evidence supports the existence of a gut-brain axis that allows bi-directional communication between the gut and brain through several pathways including neural, endocrine, and immune mechanisms11,28. Within this framework, alterations in gut microbial communities in patients with AD may result in pathophysiological changes in the brain. In support of this hypothesis, a recent study showed that transgenic AD mice raised under germ-free conditions have less cerebral amyloid deposition than conventionally-raised AD mice, indicating that gut microbiota influence the development of amyloid pathology16.

In our study, the phylum Firmicutes as a whole, as well as several families, genera, and 61 OTUs classified within Firmicutes were decreased in the AD group. A reduction in Firmicutes has been reported in the microbiome of individuals with type 2 diabetes25 as well as obesity29 (although others have reported increased Firmicutes in obesity24,30). Notably, diabetes and insulin resistance increase the risk of developing AD31,32,33. We have recently reported that insulin resistance is associated with decreased cerebral glucose metabolism and increased amyloid deposition in asymptomatic middle-aged adults enriched for risk of AD34,35. Thus, a potential mechanism by which microbial alterations in the gut may influence AD pathology is through promoting the development of insulin resistance and diabetes. While AD and Control groups did not differ with respect to diabetes prevalence (Table 1), sub-clinical differences in insulin or glucose metabolism cannot be ruled out. Further investigation will be needed to explore the relationship between microbiota and insulin resistance in AD.

In participants with AD, we observed an increase in the phylum Bacteroidetes, which was reflected by increased Bacteroidaceae at the family level, and increased Bacteroides at the OTU and genus level. The phylum Bacteroidetes encompasses a diverse and abundant group of gram-negative commensal bacteria in the gut36, including the genus Bacteroides, which has been detected at higher levels in the gut of individuals with type 2 diabetes25 and in patients with Parkinson’s disease13, a neurodegenerative disorder. The major outer membrane component of gram-negative bacteria is lipopolysaccharide (LPS), which is capable of triggering systemic inflammation and the release of pro-inflammatory cytokines after translocation from the gut to systemic circulation37. Additionally, in vitro and in vivo studies have demonstrated an association between bacterial endotoxins (e.g. LPS) and AD pathology. Co-incubation of Aβ peptide with LPS potentiates amyloid fibrillogenesis38, and systemic injection of LPS in wild-type and transgenic AD mice results in greater amyloid deposition and tau pathology39,40,41,42. In humans, intestinal permeability increases with age43, and elderly individuals show an association between increased LPS-binding protein (a marker of microbial translocation) and inflammation44. Moreover, a recent study involving postmortem brain tissue from patients with AD showed that LPS and gram-negative E. coli fragments co-localize with amyloid plaques45. Thus, increased abundance of gram-negative intestinal bacteria such as Bacteroides in participants with AD may result in increased translocation of LPS from the gut to systemic circulation, which in turn may contribute to or exacerbate AD pathology through inflammation or other mechanisms.

Additionally, compared to control participants, AD participants in our study exhibited decreased Actinobacteria. These differences were mostly driven by changes in Bifidobacterium. Actinobacteria, particularly members of the Bifidobacterium genus, are an important bacterial inhabitant of the human gut across the lifespan, and their beneficial health effects have been well-documented46,47. In particular, certain species of Bifidobacterium are associated with anti-inflammatory properties and decreased intestinal permeability48. Additionally, supplementation with Bifidobacterium has been shown to decrease LPS levels in the intestine and improve gut mucosal barrier properties in mice49,50. Interestingly, in germ-free mice colonized with human gut microbiota, increased levels of Bifidobacterium are associated with decreased bacterial translocation to systemic circulation, while increased levels of Bacteroides have been shown to increase bacterial translocation51. Considering our present findings, increased Bacteroides and decreased Bifidobacterium in AD participants may represent a gut microbial phenotype with particular propensity for translocation of pro-inflammatory bacterial components. Furthermore, several Bifidobacterium species are widely used as probiotics. A small study of probiotics that included Bifidobacterium demonstrated a change in Mini-Mental State Examination scores after a 12-week intervention among participants with severe dementia52. Taken together with the decreased abundance of Bifidobacterium in AD participants observed in our study, larger trials may be warranted, particularly in earlier disease stages.

Finally, we observed correlations between levels of differentially abundant gut microbiota and CSF biomarkers of AD pathology in a subset of participants that had also undergone lumbar puncture. In general, genera identified as more abundant in AD were associated with greater AD pathology while genera identified as less abundant in AD were associated with less AD pathology. These effects were most prominent when examining CSF p-tau/Aβ 42 , a composite measure of AD pathology. Interestingly, even among non-demented participants who had undergone lumbar puncture, we found a relationship between genera that were either more or less abundant in AD and markers of amyloid and tau protein, even in the absence of dementia. In particular, Dialister and SMB53 showed the strongest correlations in non-demented participants, with greater abundance of these bacteria associated with less AD pathology, suggesting these bacterial taxa may be protective against development or progression of AD pathology. We also observed significant associations in AD participants between CSF YKL-40 and abundance of Bacteroides, Turicibacter, and SMB53 (family Clostridiaceae). While these findings support a link between altered gut bacterial abundance and glial activation in AD, this relationship is less clear in healthy non-demented individuals and requires further investigation.

A limited number of studies have attempted to address the role of gut microbiota in AD. A recent investigation in cognitively-impaired older adults (without an AD diagnosis) reported increased abundance of the pro-inflammatory bacteria Escherichia/Shigella and decreased abundance of the anti-inflammatory bacteria Eubacterium rectale in individuals with evidence of amyloid deposition on PET imaging compared to individuals who were amyloid negative14. While those results support a link between gut microbiota and brain amyloidosis, the study only investigated the abundance of six pre-selected bacterial taxa using quantitative PCR rather than the broader approach used here. Additionally, in a recent AD mouse microbiome study using 16S rRNA sequencing, APP/PS1 transgenic mice showed increased Helicobacteraceae and Desulfovibrionaceae at the family level, increased Odoribacter and Helicobacter, and decreased Prevotella compared to wild-type mice53. However, while the anatomy and physiology of the gastrointestinal tract of humans and mice share many characteristics54, there are also substantial differences with respect to resident bacterial communities30, which makes comparing taxa and changes in abundance between these studies difficult.

While AD participants were well-matched to our Control participants (suggesting that the gut microbiome differences we observed were not likely the result of age, sex, BMI, or dietary differences between groups), they did differ with respect to the use of selective serotonin reuptake inhibitors (SSRIs) and AD medications. We did not find differences in microbial richness, diversity, or relative abundance of the 13 genera identified as altered in AD between AD participants taking SSRIs and AD participants not taking SSRIs (Supplementary Table S5), suggesting that these medications are not influencing our results. Nearly all AD participants in our study were taking the AD medications donepezil or rivastigmine (acetylcholinesterase inhibitors), and/or memantine (an NMDA receptor antagonist). It is unknown how these medications affect the gut microbiome. The most common side effects reported for acetylcholine esterase inhibitors are gastrointestinal upset55, both nausea and diarrhea, which could influence microbiota composition. It is worth noting that our participants did not report chronic constipation or diarrhea, and there was no difference between groups on the Bristol stool scale (Table 1), which can be used as a surrogate for stool transit time. Still, we recognize that we cannot completely rule out the effect of AD medication use on our results. Further work, including animal experiments and longitudinal human studies, will be needed to determine the cause-effect relationship between gut microbiota and pathogenesis of AD. Determining the role of gut bacteria in the progression or maintenance of AD may lead to novel interventional approaches that alter or restore healthy gut bacterial composition, or identification of microbial metabolites that are protective against AD.