Gut microbiome dysbiosis in Alzheimer’s disease and mild cognitive impairment: A systematic review and meta-analysis

Background Alzheimer’s disease (AD) is a neurodegenerative disorder that causes gradual memory loss. AD and its prodromal stage of mild cognitive impairment (MCI) are marked by significant gut microbiome perturbations, also known as gut dysbiosis. However, the direction and extent of gut dysbiosis have not been elucidated. Therefore, we performed a meta-analysis and systematic review of 16S gut microbiome studies to gain insights into gut dysbiosis in AD and MCI. Methods We searched MEDLINE, Scopus, EMBASE, EBSCO, and Cochrane for AD gut microbiome studies published between Jan 1, 2010 and Mar 31, 2022. This study has two outcomes: primary and secondary. The primary outcomes explored the changes in α-diversity and relative abundance of microbial taxa, which were analyzed using a variance-weighted random-effects model. The secondary outcomes focused on qualitatively summarized β-diversity ordination and linear discriminant analysis effect sizes. The risk of bias was assessed using a methodology appropriate for the included case-control studies. The geographic cohorts’ heterogeneity was examined using subgroup meta-analyses if sufficient studies reported the outcome. The study protocol has been registered with PROSPERO (CRD42022328141). Findings Seventeen studies with 679 AD and MCI patients and 632 controls were identified and analyzed. The cohort is 61.9% female with a mean age of 71.3±6.9 years. The meta-analysis shows an overall decrease in species richness in the AD gut microbiome. However, the phylum Bacteroides is consistently higher in US cohorts (standardised mean difference [SMD] 0.75, 95% confidence interval [CI] 0.37 to 1.13, p < 0.01) and lower in Chinese cohorts (SMD -0.79, 95% CI -1.32 to -0.25, p < 0.01). Moreover, the Phascolarctobacterium genus is shown to increase significantly, but only during the MCI stage. Discussion Notwithstanding possible confounding from polypharmacy, our findings show the relevance of diet and lifestyle in AD pathophysiology. Our study presents evidence for region-specific changes in abundance of Bacteroides, a major constituent of the microbiome. Moreover, the increase in Phascolarctobacterium and the decrease in Bacteroides in MCI subjects shows that gut microbiome dysbiosis is initiated in the prodromal stage. Therefore, studies of the gut microbiome can facilitate early diagnosis and intervention in Alzheimer’s disease and perhaps other neurodegenerative disorders.


Methods
We searched MEDLINE, Scopus, EMBASE, EBSCO, and Cochrane for AD gut microbiome studies published between Jan 1, 2010 and Mar 31, 2022. This study has two outcomes: primary and secondary. The primary outcomes explored the changes in α-diversity and relative abundance of microbial taxa, which were analyzed using a variance-weighted randomeffects model. The secondary outcomes focused on qualitatively summarized β-diversity ordination and linear discriminant analysis effect sizes. The risk of bias was assessed using a methodology appropriate for the included case-control studies. The geographic cohorts' heterogeneity was examined using subgroup meta-analyses if sufficient studies reported the outcome. The study protocol has been registered with PROSPERO (CRD42022328141).

Findings
Seventeen studies with 679 AD and MCI patients and 632 controls were identified and analyzed. The cohort is 61.9% female with a mean age of 71.3±6.9 years. The meta-analysis shows an overall decrease in species richness in the AD gut microbiome. However, the phylum Bacteroides is consistently higher in US cohorts (

Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a gradual loss of cognition and memory. It is expected that 78 million older adults will be diagnosed with AD by 2030 [1]. AD is preceded by a prodromal or early stage in which patients suffer mild cognitive impairment (MCI) [1]. While there is no known cure for AD, a few studies have reported success in improving cognition with non-drug interventions such as fecal microbial transplants [2,3] and probiotics in early stages [4]. One possible route of intervention for Alzheimer's may be the gut microbiome, an ecosystem of about 100 trillion commensal microorganisms representing a distinct group of 500-1000 species [5]. The importance of the gut microbiome in metabolite secretion, pathobiont restriction, and immune system maturation is well-known [5]. The gut microbiome primarily influences neurological function through the gut-brain axis, a channel of communication between the brain and the abdominal organs, through the nervous system and neuromodulator production [6].
Perturbations in gut microbiome composition, termed dysbiosis, have been linked to several diseases [7]. In neurodegenerative disorders, the pathway between gut dysbiosis and neurodegeneration includes immune activation through a defective gut barrier, induction of a systemic inflammatory response, impairment of the blood-brain barrier, and neuroinflammation [7]. Case-control studies of AD identify significant changes in microbial composition, with a greater abundance of pro-inflammatory bacterial genera such as Escherichia-Shigella, and a decrease in anti-inflammatory species such as E. rectale [8]. However, till date and to the best of our knowledge, no specific microbial taxa have been consistently and uniquely associated with Alzheimer's. Furthermore, case-control studies with smaller samples may be affected by low power and confounding factors which obscure true biological signals. With several case-control studies being published in recent years, it seems an appropriate next step to pool the studies together in a meta-analysis to derive robust insights with potential for clinical impact.
In this work, we systematically reviewed case-control studies of AD and MCI patient gut microbiomes. The outcomes of gut microbiome studies namely, changes in α-diversity, βdiversity, changes in relative abundance, and linear discriminant analysis effect sizes, were studied to identify the microbial taxa consistently impacted by gut dysbiosis. Growing evidence relating gut dysbiosis to Alzheimer's and other forms of dementia emphasizes the timely nature of this article.

Materials and methods
The study protocol for this systematic review and meta-analysis was finalized in advance of data collection and has been registered with The International Prospective Register of Systematic Reviews (PROSPERO), number CRD42022328141 (accessible at https://www.crd.york.ac. uk/PROSPERO/display_record.php?RecordID=328141). The protocol was written according to PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) guidelines. A PRISMA Protocols (PRISMA-P) checklist of items addressed in the systematic review protocol can be found in S1 Table.

Search strategy
We searched MEDLINE, Cochrane, EBSCO, EMBASE, and Scopus, for case-control metagenomic and 16S studies of Alzheimer's disease (AD) and mild cognitive impairment (MCI) in humans. The search was limited to papers written in English and published between Jan 1, 2010, and Mar 31, 2022. A set of controlled vocabulary terms related to Alzheimer's and metagenomics were formulated and combined with the 'AND' operator to generate search queries. The controlled vocabulary has been provided in S2 Table.

Inclusion and exclusion criteria
Studies were eligible if they assessed the gut microbiomes of human patients with AD or MCI with metagenomic sequencing and reported outcomes such as α-diversity, β-diversity ordination, relative abundances of various taxa, and linear discriminant analysis effect sizes (LEfSe). Eligible study designs were case-control and intervention studies (with baseline sampling). Patients had to meet well-defined diagnostic criteria such as DSM (Diagnostic and Statistical Manual of Mental Disorders) or NIA-AA (National Institute on Aging and Alzheimer's Association) guidelines. If patients self-reported decreases in cognition and memory (corroborated by a caregiver) without meeting the diagnostic criteria for AD, they were considered to have MCI. Controls were cognitively normal (CN) subjects, reasonably matched to the AD and MCI cohorts in age, gender, years of education, and lifestyle. Exclusion criteria included antibiotic use within two weeks of sample collection and the presence of confounding conditions such as depression, cancer, or any other genetic/neurological disorders.

Study selection and data extraction
All identified records were imported into Rayyan, a widely-used mobile and web application that helps expedite the initial screening of records for systematic review [9], for de-duplication and screening. De-duplication was automatic if records were matched 100% and manually performed if the similarity ranged from 80-99%. Titles and abstracts were initially screened for eligibility by two authors. Full-text reports of selected studies were further assessed using the selection criteria by two authors, with a plan to resolve any disagreements by the corresponding author. We also performed manual searches of the reference lists of the included studies. Reported data in tabular and graphical form was extracted, cleaned, and tabulated from the full-text reports. The corresponding authors of included studies were contacted to request any missing data.

Outcome measures
The primary outcomes of interest were measures of microbial diversity, namely changes in αdiversity and relative abundance of various taxa. Microbial diversity refers to the variety (richness) and abundance (evenness) of species in a defined unit of study [10,11]. In this study, αdiversity outcomes such as Shannon, Simpson, Chao index, Abundance-based Coverage Estimators (ACE), and the number of species observed (S obs ) were included. The relative abundances (proportion of a given taxon) at the phylum, family, and genus levels were also examined.
Secondary outcomes of interest included β-diversity ordination and LEfSe results. The results of ordination (summarization of distance matrix and projection in a low-dimension space) and statistical testing of β-diversity indices (Bray-Curtis, Jaccard, weighted/unweighted UniFrac, Aitchison distances) were examined. The definitions and interpretation of the diversity indices have been provided in S1 Appendix. Additionally, we included a qualitative analysis of LEfSe (linear discriminant analysis effect size) [12]. LEfSe identifies differentially abundant taxa between two groups of metagenomic samples using non-parametric statistical tests and linear discriminant analysis with strict cut-offs.
Where necessary, units were converted so that related outcomes were on consistent scales (for instance, percentage composition was converted to proportion).

Additional data items
In addition to reported primary and secondary outcomes, the following data were recorded: study location, cohort size, the average age of the cohort, the proportion of female participants, diagnostic and exclusion criteria used for patients, sequencing platform, bioinformatics methods for data analysis, and details of ethical committee/review board approvals.

Statistical analysis
All statistical analyses were performed using the R programming language (version 4.0.3, The R Foundation for Statistical Computing, Vienna, Austria). The R metaphor package was used for the quantitative synthesis of α-diversity and relative abundance data. LEfSe results and βdiversity outcomes were summarized qualitatively. For quantitative synthesis, continuous outcomes were reported using the standard mean difference (SMD). Three or more studies measuring the same outcome were combined with an inverse-variance weighted random-effects model. Reported median and interquartile range values were converted to estimates of the mean and standard deviation (SD) [13]. Means with 95% CIs were converted to means and SDs using the formula provided by the Cochrane Handbook. Forest plots were generated for each primary outcome of interest, with the weight indicating the influence of an individual study on the pooled result. Heterogeneity was quantified using τ 2 (tau 2 ) and I 2 statistics. We examined the significance of heterogeneity using the χ 2 (chi 2 ) test. A P value less than 0.05 was considered statistically significant. If significant heterogeneity was present, we performed a subgroup meta-analysis by grouping studies according to sample characteristics, provided that sufficient studies reported the outcome. A priori variables identified for the subgroup analysis were age, sex and study location. did not formally assess the quality of the included studies, as the outcome data was generated from next-generation sequencing methods and analyzed using standard bioinformatics workflows such as QIIME.

Results
Of 2235 records identified, 42 full-text articles were screened, of which 17 publications of 17 unique studies were deemed eligible and included in the narrative synthesis (S3 Table). The study selection process has been summarized in Fig 1. All included studies were deemed to provide sufficient data for inclusion for our study. An overview of the characteristics of each study is in S4 Table. All 17 included studies identify significant alterations in gut microbiome composition of AD or MCI patients compared to CN subjects.
The 17 included studies comprise 679 patients (241 with MCI and 438 with AD), and 632 controls. The cohorts were 61.9% female with a mean age of 71.3±6.9 years. The cohorts in the included studies ranged in size from 11 to 46 MCI patients and 7 to 100 AD patients. The corresponding controls (cognitively normal or CN group) were matched with the MCI and AD patients in terms of age and proportion of female participants. The controls also led a lifestyle similar to the MCI and AD patients. All studies have used well-defined diagnostic criteria such as DSM and NIA-AA guidelines for AD/MCI patient inclusion. Twelve studies analyzed AD cohorts and ten studies analyzed MCI cohorts. Most studies were undertaken in China (n = 11; 65%). Two studies were randomized controlled trials [15,16], and two were derived
Most included studies used the Illumina MiSeq platform to sequence V3-V4 regions of bacterial 16S rRNA. Exceptions include Pan et al. [26] who sequenced the V1-V9 region, and Nagpal et al. [16] and Vogt et al. [27], who sequenced the V4 region. Haran et al. [17] performed shotgun sequencing with Illumina NextSeq 500, and therefore, their data were included in the synthesis of β-diversity outcomes. Although there were some heterogeneity in the bioinformatic analyses, the introduction of QIIME (Quantitative Insights Into Microbial Ecology) software [32] has provided scientists with a uniform framework for the analysis of 16S data. The majority of the included studies have employed QIIME. The bioinformatics methods and taxonomic units used for each included study in have been summarized in S5 Table. α-diversity Shannon and Simpson's indices are indicative of both richness and evenness. Nine studies reported the Shannon index for AD patients [18, 20-24, 27, 28, 30]. Overall, a small, significant decrease was observed in Shannon diversity (SMD -0.23, 95% CI -0.5 to 0.05; p = 0.05; Fig 2A) with substantial, significant heterogeneity (I 2 = 60%; p < 0.01). A subgroup meta-analysis by location showed a small, significant reduction in Chinese cohorts (SMD -0.28, 95% CI -0.51 to -0.06; p = 0.01), with low heterogeneity (I 2 = 40%; p = 0.12). We also observed a small, insignificant increase in the Shannon diversity of MCI patients, irrespective of location (SMD 0.13, 95% CI -0.07 to 0.33; p = 0.17, Fig 3A). The meta-analysis of the Simpson index is provided in the Extended Results S2 Appendix.
The Actinobacteria and Proteobacteria phyla represent 10% of the gut microbiome. The aging gut is marked by an increase in Proteobacteria [35], a highly heterogenous taxon of facultative anaerobes. Four studies have reported the relative abundance of Proteobacteria in AD and MCI patients. In AD patients, the standardized mean differences of individual studies ranged from -0.62 to 0.75. Overall, no significant effect was observed (SMD 0.05, CI -0.63 to 0.73; p = 0.89; Fig 4C), but there was significant heterogeneity (I 2 = 88%; p < 0.01). In MCI patients, there was no change in the abundance of Proteobacteria (SMD 0.07, CI -0.24 to 0.38; p = 0.67; Fig 5C). In contrast, the Actinobacteria phylum showed higher relative abundance in two AD cohorts. Actinobacteria are mainly represented by Bifidobacteriaceae, and may have antidepressant effects through tryptophan production [36]. Elderly individuals have lower levels of Actinobacteria [35]. Overall, there was a moderate but insignificant increase in AD (SMD 0.63, 95% CI -0.62 to 1.89; p = 0.32; Fig 4D), with substantial, significant heterogeneity (I 2 = 97%; p < 0.01). While Li et al. (2019) [22] reported a moderate increase in Actinobacteria in MCI patients, no other studies have reported Actinobacteria abundance at the MCI stage.

Linear discriminant analysis effect sizes (LEfSe)
All nine studies which reported LEfSe to identify differentially abundant taxa between AD/ MCI and CN participants [20-24, 26, 28-30] have been included for the following qualitative summary. Key similarities and differences in the commonly-represented taxa, i.e., phyla, families, and genera, have been compiled in Table 2. LEfSe analysis uses statistical testing and modeling with a rigorous cut-off to determine the most discriminative features between the two groups. Moreover, the false positive rate was reported to be lower than 0.05. Therefore, a taxon was deemed consistent if it was reported by two or more studies to be of higher abundance (or lower abundance) in AD compared to controls, and there were no studies which report contrasting LEfSe scores for the same taxon. We have included information on the score cut-offs to reflect the strictness of the comparison.
Six studies reported LEfSe for Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. Two studies reported an increased abundance of Actinobacteria in AD [23,29] In the case of Prevotella " # # a Not available; Up arrow (") indicates the relative abundance of the taxa is higher in AD patients' microbiota. The down arrow (#) indicates lower relative abundance in AD patients' microbiota compared to cognitively normal controls. https://doi.org/10.1371/journal.pone.0285346.t002

Risk of bias assessment
The overall risk of bias assessment for all included papers has been summarized in Table 3.
Four studies showed an unclear risk of bias. One study included selected patients slated to  [19]. In another study, the elders were part of a longitudinal observation study and thus were not adequately matched [18]. Likewise, in the study by Haran et al. [17], participants were drawn from a longitudinal study of elders in care facilities. We were able to assess selective reporting bias for Shannon index and relative abundance of genus Bacteroides. The reporting bias was low according to the funnel plots from the two outcomes, provided in S1 Fig. We deemed the evidence related to primary outcomes of high quality due to the next-generation sequencing methods and established analysis pipelines used to generate the data.

Discussion
In this work, gut microbiome studies were systematically reviewed in AD and MCI to obtain insights into the direction and extent of gut dysbiosis in terms of diversity and relative abundances of various taxa. In terms of α-diversity, a slight increase was observed in MCI patients. However, in AD patients, there was a slight, significant decrease in the Shannon and Simpson indices. The latter observation is inconsistent with previous meta-analyses of α-diversity indices in Parkinson's disease [43]. Moreover, in AD patients, there were significant decreases in S obs , ACE, and the Chao index. MCI patients also showed a small, but insignificant, decrease in Chao index. Better consensus was seen in terms of β-diversity, with most studies reporting significant differences among AD, MCI and CN cohorts. Therefore, our meta-analyses of both α-and β-diversity outcomes suggest significant differences in terms of species richness and less change in evenness.
From the meta-analyses of relative abundances, there were small to moderate, mostly insignificant changes in MCI patients. Although insignificant, the Firmicutes phylum increased moderately and the Bacteroidetes phylum decreased moderately. The Bacteroides genus also shows a small, insignificant decrease. Interestingly, there was a moderate, significant increase in Phascolarctobacterium, which was also observed in AD patients but without significance. Moreover, in AD patients, there was a mild, insignificant increase in Firmicutes and Actinobacteria, a small, insignificant decrease in Bacteroidetes, and no change in Proteobacteria. At the family level, Lachnospiraceae and Bacteroidaceae decreased and Bifidobacteriaceae increased in abundance, further supported by our analysis of LEfSe. Notably, regional differences were observed in the abundance of the Bacteroides genus. Our meta-analysis revealed that Bacteroides was reduced in Chinese cohorts but showed an increase in US-based AD cohorts, thus supporting the notion that region, diet and lifestyle may have a considerable influence on the gut composition and therefore, AD pathophysiology. The LEfSe synthesis further corroborates our observation that Bacteroides was reduced in Chinese AD cohorts. Additionally, LEfSe results show an increase in Enterobacteriaceae, Lactobacillaceae, Bifidobacterium, Akkermansia, and Clostridium, and a decrease in Anaerostipes and Lachnospira.
The gut microbiota is responsible for the production of several metabolites, of which SCFAs are of particular interest for their role in gut health and inflammation. For instance, butyrate has been shown to reduce inflammation and regulate the host immune system [35]. Treatment with sodium butyrate in germ-free mice resulted in decreased blood-brain barrier (BBB) permeability [44]. However, a recent study also linked SCFA supplementation to increased amyloid-β burden [45]. Furthermore, the LEfSe synthesis has shown an increased abundance of acetate, lactate, and propionate producers such as Akkermansia, Lactobacillus and Bifidobacterium, which have been shown to correlate negatively with clinical indicators of cognitive function [23]. Additionally, they may also have pathogenic relevance [46,47]. Species-level microbiome profiling and metabolomic analyses would provide more granular insights into the role of the gut-brain axis in Alzheimer's and other dementias.
Regional differences in Bacteroides abundance in AD indicate that diet and lifestyle have a crucial role in gut microbiome composition, which subsequently affects the gut-brain axis and development of AD. Our study highlights the relevance of region-based longitudinal studies in aging cohorts. The differences in Bacteroides, and the heterogeneity of other taxa, suggest the possibility of stratifying patient cohorts as a first step to microbiome-related interventions [31]. Moreover, the extent of changes in Bacteroides, one of the major constituents of the gut microbiome, is higher in the AD stage than the MCI stage. Along with the increase in Phascolarctobacterium, this underscores the need for early diagnosis at the MCI stage. Recent studies lend support to the possibility that non-drug interventions can be particularly impactful in this stage [4,15,16].
The strengths of this study include the comprehensive set of outcomes and rigorous methodology. Nonetheless, it is recognized that the interpretation of our study is subject to possible limitations. While most studies have documented medications taken by participants, only one study has addressed polypharmacy [17], which refers to the use of five or more daily medications, and is known to be detrimental to gut microbiome composition [48]. This may represent a source of bias in addition to observed heterogeneity in the gut microbiome composition of elderly individuals [49]. It would be informative to examine the differences in microbiome composition between participants who practice polypharmacy and those who don't.

Conclusion
In conclusion, we have discovered that the progression of Alzheimer's is associated with more significant impact on species richness than evenness in the gut microbiome. We have obtained evidence that regional differences in diet and lifestyle can influence gut dysbiosis in AD patients. Our study has provided a glimpse into specific re-arrangements occur during the progression to AD. Therefore, we believe that our study will be helpful in the development of nondrug early-stage interventions which harness the power of the gut microbiome to affect the gut-brain axis.