Association of The Gut Microbiota With Cognitive Function in Midlife

Original Investigation | Neurology
Association of the Gut Microbiota With Cognitive Function in Midlife

Katie Meyer, ScD; Anju Lulla, PhD; Kunal Debroy, BS; James M. Shikany, DrPH; Kristine Yaffe, MD; Osorio Meirelles, PhD; Lenore J. Launer, PhD
Abstract Key Points

Question Is the gut microbiota
IMPORTANCE Animal experiments and small clinical studies support a role for the gut microbiota in
associated with cognitive function?
cognitive functioning. Few studies have investigated gut microbiota and cognition in large
community samples. Findings In this cross-sectional study,
β-diversity, a measure of gut microbial
OBJECTIVE To examine associations of gut microbial composition with measures of cognition in an community composition, was
established population-based study of middle-aged adults. statistically significantly associated with
all measures of cognitive function.
DESIGN, SETTING, AND PARTICIPANTS This cross-sectional study analyzed data from the Several specific genera were also
prospective Coronary Artery Risk Development in Young Adults (CARDIA) cohort in 4 US significantly associated with 1 or more
metropolitan centers between 2015 and 2016. Data were analyzed in 2019 and 2020. measures of cognitive function after
adjustment for multiple comparisons.
EXPOSURES Stool DNA were sequenced, and the following gut microbial measures were gathered:
Meaning These results are consistent
(1) β-diversity (between-person) derived with multivariate principal coordinates analysis; (2)
with an association between the gut
α-diversity (within-person), defined as richness (genera count) and the Shannon index (integrative
microbiota and cognitive function, and
measure of genera richness and evenness); and (3) taxonomy (107 genera, after filtering).
support further mechanistic and
population work to elucidate the
MAIN OUTCOMES AND MEASURES Cognitive status was assessed using 6 clinic-administered
potential for gut microbiota targets for
cognitive tests: Montreal Cognitive Assessment (MoCA), Digit Symbol Substitution Test (DSST),
prevention or treatment of
Rey-Auditory Verbal Learning Test (RAVLT), Stroop, category fluency, and letter fluency. A global
cognitive decline.
score measure derived using principal components analysis was also assessed; the first principal
component explained 56% of variability.
+ Supplemental content
RESULTS Microbiome data were available on 597 CARDIA participants; mean (SD) age was 55.2 (3.5) Author affiliations and article information are
years, 268 participants (44.7%) were men, and 270 (45.2%) were Black. In multivariable-adjusted listed at the end of this article.
principal coordinates analysis, permutational multivariate analysis of variance tests for β-diversity
were statistically significant for all cognition measures (principal component analysis, P = .001;
MoCA, P = .001; DSST, P = .001; RAVLT, P = .001; Stroop, P = .007; category fluency, P = .001) with
the exception of letter fluency (P = .07). After adjusting for sociodemographic variables (age, race,
sex, education), health behaviors (physical activity, diet, smoking, medication use), and clinical
covariates (body mass index, diabetes, hypertension), Barnesiella was positively associated with the
first principal component (β, 0.16; 95% CI, 0.08-0.24), DSST (β, 1.18; 95% CI, 0.35-2.00), and
category fluency (β, 0.59; 95% CI, 0.31-0.87); Lachnospiraceae FCS020 group was positively
associated with DSST (β, 2.67; 95% CI, 1.10-4.23), and Sutterella was negatively associated with
MoCA (β, −0.27; 95% CI, −0.44 to −0.11).
CONCLUSIONS AND RELEVANCE In this cross-sectional study, microbial community composition,

based on β-diversity, was associated with all cognitive measures in multivariable-adjusted analysis.
These data contribute to a growing body of literature suggesting that the gut microbiota may be
associated with cognitive aging, but must be replicated in larger samples and further researched to
identify relevant pathways.
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Open Access. This is an open access article distributed under the terms of the CC-BY License.
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JAMA Network Open | Neurology Association of Gut Microbiota With Cognitive Function in Midlife
Introduction
Communication pathways between gut bacteria and neurologic function (referred to as the “gut-
brain axis”) have emerged as a novel area of research into potential mechanisms regulating brain
health1,2 through immunologic, metabolic, and endocrine pathways.3,4 Several studies have shown
associations between gut microbial measures and neurological outcomes, including cognitive
function and dementia. Mechanisms have not been fully established, but there is growing support for
a role in microbiota-generated short-chain fatty acids.5,6 In animal experiments, germ-free or
antibiotic-treated rodents, which have reduced microbial diversity, have been shown to display
cognitive deficits, such as reduced memory, impaired working memory, and changes in brain-derived
neurotrophic factor in the hippocampus.1,2,7,8 Small-scale human studies have shown associations
between microbial features and cognition, or found significant improvements when comparing
controls with persons who have been treated with probiotics to increase commensal microbiota.1
However, few community-based studies have been conducted with large and diverse populations.
In this study we examine cross-sectional associations of gut microbial diversity and taxonomic
composition with cognitive status among middle-aged adults recruited from 4 US centers in the
Coronary Artery Risk Development in Young Adults (CARDIA) study. We hypothesized that: (1) gut
microbial diversity is positively associated with global and domain-specific cognitive status, and (2)
higher cognitive status is associated with specific taxonomic groups that are involved in short-chain
fatty acid production. Given our current lack of understanding of the potential role for the microbiota
in cognitive functioning, we have applied multiple-comparisons adjustment on the full set of tested
genera, rather than a subset of genera related to short-chain fatty acid production.
Methods
Study Sample
CARDIA is a population-based, well-characterized, and sociodemographically diverse study of
community-dwelling Black and White adults living in 4 metropolitan areas: Birmingham, Alabama;
Chicago, Illinois; Minneapolis, Minnesota; and Oakland, California.9 At CARDIA’s Year 30 follow-up
examination in 2015-16 (age range, 48-60 years; 3358 participants [70.9%] from the surviving
cohort), all participants were offered a battery of cognitive assessments as part of an ancillary study,
with 3124 (93.0%) completing at least 1 assessment. In addition, 615 Year 30 participants (18.3%)
were recruited into a microbiome substudy, details of which have been previously reported.10
Analyses for the present study were conducted in 2019 and 2020. All CARDIA field centers received
institutional review board approvals (University of Minnesota, University of Alabama at Birmingham,
Northwestern University, and Kaiser Permanente Northern California Division of Research) and
participants provided written informed consent to all study components. This study followed the
Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting
guideline.
Microbiome Data
Standard protocols were followed for collection and processing of stool samples11,12 as previously
described.10 Participants in the microbiome study completed the stool collection in their home.
Participants shipped their samples to the Nutrition Research Institute at the University of North
Carolina, Chapel Hill, where samples were stored at −80 °C until processing. Samples were collected
in tubes containing a stabilizing solution (Thermo Fisher Scientific) and shipped overnight in
insulated containers with gel ice packs. DNA was extracted from 0.2 g of stool using a DNA isolation
kit (Qiagen). The V3 and V4 hypervariable regions were amplified and sequenced using a sequencer
platform (2 × 300 bp) (Illumina), yielding a median (IQR) 165 536 reads (144 058-209 556 reads).
Forward sequences were processed (for quality trimming, denoising, and chimera removal) through
the divisive amplicon denoising algorithm DADA2 package in R (R Project for Statistical Computing)13

for a postprocessing median (IQR) of 115 869 reads (98 550-139 706). The DADA2-formatted Silva
database was used to assign taxonomy.14
Cognitive Assessments
Cognitive function was introduced in CARDIA at the Year 25 (2010-2011) examination. At the Year 30
follow-up, participants were administered 6 cognitive tests: the Digit Symbol Substitution Test
(DSST), Rey-Auditory Verbal Learning Test (RAVLT); the timed Stroop test, letter fluency, and
category fluency; and the Montreal Cognitive Assessment (MoCA) (scales provided in eTable 2 in the
Supplement). Higher scores reflect better performance on all assessments except Stroop, for which
higher scores reflect poorer performance.
Covariates and Confounders

We included in our analyses covariates that may confound associations between cognitive test scores
and microbiome characteristics. Standard questionnaires were used to obtain demographic and
health behavior data at the CARDIA field centers during core examinations. CARDIA participants
reported their sex and race at baseline, and age and educational attainment at each examination.
Black and White race were self-reported by participants at the baseline examination—we adjusted for
race in the analysis to reflect differences in both microbiome and cognition measures by race but did
not hypothesize differences in associations. Educational attainment was calculated as the maximum
(in years) reported by participants over their 30-year examination history. At each examination,
participants reported their use of tobacco products and completed an interviewer-administered
CARDIA Physical Activity Questionnaire, from which a total activity score was calculated.15 A brief
diet assessment was completed by microbiome study participants, from which we derived a
summary measure of diet quality as previously reported in CARDIA16 with higher scores indicating
higher diet quality. Participants reported their use of medications, including for hypertension,
elevated lipids, and diabetes. A medication use score was created as the total number of medications
reported by participants.
Standardized protocols were used by trained staff for all clinical measures. Height and weight
were measured to the nearest 0.5 cm and 0.2 kg, respectively, for body mass index (BMI; calculated
as weight in kilograms divided by height in meters squared). Resting systolic and diastolic blood
pressure (BP) measures were taken in the seated position. BP values were calculated as the mean of
the second and third of 3 measurements taken with oscillometer calibrated to a random-zero
sphygmomanometer. Hypertension was defined, based on the clinical standard at the time of the
Year 30 examination, as current use of antihypertensive medication, a systolic BP 140 mm Hg or
higher, or a diastolic BP 90 mm Hg or higher. Diabetes was defined as having fasting glucose 126
mg/dL (to convert to millimoles per liter, multiply by 0.0555) or greater, 2-hour oral glucose tolerance
test 200 mg/dL or greater, hemoglobin A1c 6.5% (to convert to proportion of total hemoglobin,
multiply by 0.01) or greater, or the use of hypoglycemic medications.
Analytical Sample
Of 615 participants who enrolled in the microbiome study, 607 had viable stool DNA for sequencing.
Ten participants were missing data on all cognitive tests, yielding an analytic sample of 597.
Participants with missing data on specific cognitive tests were excluded for analysis of those
respective tests (596 participants for MoCA, 591 for letter fluency, and 589 for Stroop). Participants
with additional missing data were excluded from regression models on a covariate basis (3 for
physical activity, 7 smoking, 46 diet, and 4 diabetes), yielding sample sizes ranging from 537 to 597
depending on the level of covariate adjustment.
Statistical Analysis
Statistical analysis was conducted on 3 standard microbial measures: within-person α-diversity,
between-person β-diversity, and individual taxa. Primary analysis was at the genus level, the most

refined taxonomy from these data. The R package vegan was used to generate measures of microbial
diversity.17 Measures of α-diversity were derived from raw counts and included richness (the rarified
number of genera) and Shannon Diversity Index, a function of both richness and evenness.18
Richness was calculated as the number of distinct genera per participant, with total per-participant
abundance rarified through random sampling to the minimum count across all participants. We used
multivariate principal coordinates analysis (PCoA) to assess β-diversity in microbial community
composition.19
Prior to β-diversity analysis, raw genera counts were log-transformed as previously described.20
PCoA was used to generate orthogonal measures of microbial composition based on a distance
matrix of microbial abundance (Bray-Curtis). For regression analysis of distinct taxa, we removed
genera with a count of zero in at least 25% of participants to limit the influence of rare taxa and the
possibility of spurious findings.10 After this filtering, 107 of 375 originally assigned genera (28.5%)
remained for analysis.
We generated a summary cognitive measure based on a principal component analysis (PCA) of
the 6 assessments. Each assessment was standardized as normal-inverse scores (mean [SD], 0 [1]).
To limit exclusions from the PCA because of missing data, we ran PCA on the sample with complete
cognitive assessment data (583 participants), set missing cognitive assessment data to zero, and
applied factor loadings to nonmissing normal-inverse scores in the remaining sample. Statistical
modeling focused on the first factor from PCA (first principal component [PC]), which explained 56%
of variance and had loadings of 0.44 for MoCA, 0.43 for RAVLT, 0.40 for DSST, 0.40 for letter fluency,
0.39 for category fluency, and −0.38 for Stroop.
Associations between α-diversity and cognition measures were tested with multivariable-
adjusted regression. Differences in β-diversity were tested with permutational multivariate analysis
of variance (PERMANOVA). We conducted separate multivariable-adjusted linear regression models
for each genus, adjusting for multiple comparisons using the Benjamini-Hochberg method for false
discovery rate (FDR).21 We considered an FDR-adjusted P < .20 in 2-sided tests as significant.
Multivariable-adjusted analysis controlled for covariates that may have confounded
associations between microbial and cognitive measures, including variables that have been
associated with cognition in CARDIA or other studies. We note that some covariates may reflect
mediators in associations between the microbiome and cognition, such as BMI and diabetes.
Covariates included in sequential modeling were sequencing batch, age, sex, race, field center,
education, physical activity, current smoking, diet quality, number of medications, BMI,
hypertension, and diabetes. Statistical interaction by race or sex was tested by including
crossproduct term in regression models.
Results
Participant characteristics are shown in the Table. Participants were aged between 48 and 60 years
(mean (SD) age, 55.2 [3.5] years), 267 (44.7%) were men, 270 (45.2%) were Black, and 268 (44.8%)
were White. Compared with those not included in the microbiome study, participants in the
microbiome study had lower mean BMI, were less likely to have prevalent hypertension, and
performed slightly better on cognitive tests (eTable 1 in the Supplement). Distributions of cognitive
function measures are shown in eTable 2 in the Supplement. Correlations among cognitive
assessments ranged from −0.36 between category fluency and Stroop, to 0.64 between MoCA and
RAVLT (eTable 3 in the Supplement). Descriptive statistics for genera included in the analysis are
shown in eTable 4 in the Supplement.
Overall Microbial Community Composition

Descriptive data for cognitive assessments and genera with respect to the first 2 PCoA axes are
provided in eTables 5 and 6 in the Supplement, respectively. Cognitive function scores were
significantly associated with β-diversity based on PCoA (PCA, P = .001; MoCA, P = .001; DSST,

P = .001; RAVLT, P = .001; Stroop, P = .007; category fluency, P = .001) with the exception of letter
fluency (P = .07) (eFigure in the Supplement). We observed a statistically significant PERMANOVA
test for interaction by sex with RAVLT (P = .04) (eTable 8 in the Supplement), although PCoA
differences were significant in both men and women (eTable 9 in the Supplement). PERMANOVA
tests for interaction by race were not significant (eTables 8 and 9 in the Supplement).
α-Diversity
In multivariable-adjusted analysis, Shannon diversity was positively associated with DSST (β, 1.27;
95% CI, 0.05-2.48), indicating a 1.27 higher DSST score (by SD-units) for an SD-unit higher Shannon
diversity index score. Other findings for α-diversity and cognition were not significant (eTable 5 in the
Supplement). There was no evidence for interaction by race or sex (eTables 10 and 11 in the
Supplement).
Table. Participant Characteristics for Individuals Who Completed

Cognitive Assessments at Year 30 (2015-2016): CARDIA
Microbiome Study
Characteristic No. (%) (N = 597)a

Age, mean (SD), y 55.2 (3.5)
Sex
Men 267 (44.7)
Women 330 (55.3)
Race
Black 270 (45.2)
White 327 (54.8)
Education, mean (SD), y 15.9 (2.6)
Field center
Birmingham, Alabama 96 (16.1)
Chicago, Illinois 292 (48.9)
Minneapolis, Minnesota 108 (18.1)
Oakland, California 101 (16.9)
Current smoking 77 (13.1)
Physical activity, median (IQR), intensity unitsb 269 (127-504)
BMI, mean (SD) 29.4 (6.2)
Hypertension 209 (35.0)
Diabetes 91 (15.4)
Cognitive assessments, median (IQR)c
MoCA 25 (22-27)
DSST, No. correctly substituted 70 (58-82)
Stroop, No. correctly assigned 20 (15-27)
RAVLT, No. of words recalled 9.4 (8-10.6)
Category fluency, No. of unique animals 20 (17-24)
Letter fluency, No. unique words 42 (34-50)
Abbreviations: BMI, body mass index (calculated as weight in kilograms divided

by height in meters squared); DSST, the Digit Symbol Substitution Test; MoCA,
Montreal Cognitive Assessment; RAVLT, Rey-Auditory Verbal Learning Test.
a
Sample size represents the number of participants for whom a microbiome
sample and at least 1 cognitive assessment was present.
b
Physical activity intensity units were generated from data on the frequency,
duration, and intensity for 13 activities.
c
See eTable 2 in the Supplement for scoring scales. Mean (SD) for cognitive
measures were: MoCA, 24.4 (3.6); DSST, 69.2 (16.9); Stroop, 22.2 (11.5); RAVLT,
9.3 (1.9); category fluency, 20.6 (5.2); letter fluency, 42.6 (12.3).

Distinct Taxonomies
Accounting for multiple comparisons, genera-specific analysis revealed several associations with
cognitive assessments, but few associations remained significant (at the FDR-adjusted threshold of
P < .20) in fully adjusted models (eTables 12-18 in the Supplement). Assessments were associated
with genera at varying levels of multivariable adjustment (FDR-adjusted P < .20) (Figure). In these
models, β-coefficients indicate the difference in the cognitive assessment measures (see eTable 2 in
the Supplement for distributional data) associated with a 1-unit higher log-transformed genera
abundance. In fully adjusted analysis controlling for sociodemographics, health behaviors, and
clinical measures, Barnesiella (median [IQR] log-transform, 0.82 [0-3.02]) was positively associated
with DSST (β, 1.18; 95% CI, 0.35-2.00), category fluency (β, 0.59; 95% CI, 0.31-0.87), and the first PC
(β, 0.16; 95% CI, 0.08-0.24); Lachnospiraceae FCS020 group (median (IQR) log-transform, 1.38
[0-1.75]) was positively associated with DSST (β, 2.67; 95% CI, 1.10-4.23); Akkermansia (median [IQR]
log-transform, 1.57 [0-3.00]) was positively associated with DSST (β, 1.28; 95% CI, 0.39-2.17), and
Sutterella (median [IQR] log-transform, 2.80 [0-3.37]) was negatively associated with MoCA (β,
−0.27; 95% CI, −0.44 to −0.11). Nine genera were associated with more than 1 cognitive test in
sociodemographics-adjusted analysis (without education), including 6 from within Clostridia class (3
of which were from Lachnospirales order). We observed significant statistical tests for interaction by
race or sex for several genera, but a large number of tests were conducted, even with
FDR-adjustment, and there was a relatively small sample size (eTables 19-60 in the Supplement).
In secondary analysis of phyla (descriptive data available in eTable 61 in the Supplement),
Verrucomicrobia was positively associated with DSST (β, 1.34; 95% CI, 0.43-2.26) (eTables 62-68 in
the Supplement) and Proteobacteria was positively associated with category fluency (β, 1.08; 95%
CI, 0.34-1.82). The ratio of Firmicutes to Bacteroidetes was not associated with cognitive measures in
these data (eTable 69 in the Supplement).
Discussion
In this cross-sectional analysis of data from a population-based cohort of middle-aged Black and
White US adults, gut microbial composition was associated with domain-specific and global
Figure. Multivariable-Adjusted Associations Between Specific Genera and Measures of Cognitive Function
Category Letter Positive

Genus PC DSST Stroop RAVLT MOCA
fluency fluency
Negative
Akkermansia Multivariable adjustment level 1
Barnesiella Multivariable adjustment level 2
Christensenellaceae R.7 group
Multivariable adjustment level 3
Defluviitaleaceae UCG.011
Faecalibacterium
Hungatella
Lachnoclostridium
Lachnospira
Lachnospiraceae FCS020 group
Lachnospiraceae UCG.001
Monoglobus
Parasutterella
Subdoligranulum
Sutterella
Turicibacter
Genera are restricted to those associated with at least 1 cognitive measure with an significant after adjustment for sequencing batch, age, race, sex, study center; dotted,
FDR-adjusted P < .20. Colors reflect direction of β coefficients (blue indicates positive; results remained significant after additional adjustment for education; horizontal lines,
orange, negative). Stroop is on its natural scale, with lower values reflecting higher results were significant after further adjustment for physical activity, diet, smoking,
cognitive scores. Patterning indicates the highest level of multivariable adjustment for medication use, body mass index, diabetes, and hypertension.
which results were statistically significant, as follows: solid indicates results were

measures of cognition. A multivariate measure of between-person differences in microbial

community structure was significantly associated with cognitive measures adjusted for
sociodemographics, health behaviors, and clinical risk factors. In contrast, within-person microbial
diversity was generally not associated with cognition in these data. Genera-specific associations were
sensitive to covariate adjustment, and relatively few associations remained significant in fully
adjusted models.
Our findings are consistent with results from animal models and small clinical studies. However,
direct comparisons across human studies can be difficult given the limited sample sizes of many
published reports, the use of case-control designs comparing normal controls with cases of
dementia, variation in the taxonomic level of analysis, and the inconsistent adjustment for potential
confounders.22 Findings from our genera-specific analysis are consistent with proposed pathways
through production of the short-chain fatty acid butyrate, many members of which are within class
Clostridia.23 In animal models, administration of butyrate has been shown to be protective against
vascular dementia24,25 and cognitive impairment,26,27 as well as against metabolic risk factors for
cognitive decline and dementia.28 In a study of transgenic mice, an antibiotic-induced increase in the
relative abundance of genus Lachnospiraceae, positively associated with DSST and the first PC in our
data, was accompanied by upregulation of FOXP3+ regulatory T cells and reduced deposition of β
amyloid in brain tissue.8 Akkermansia, associated with DSST in our data, is a mucin-degrading genus
that has been shown to improve gut membrane integrity,29,30 associate negatively with
inflammation and adverse metabolic outcomes,30-33 and associate positively with cognitive
function.34 Additional data from larger human studies are needed to confirm our results and test
translation of findings from animal models to human samples. We identified a potentially novel
positive association between Barnesiella and cognitive function. Barnesiella remained positively
associated with DSST, category fluency, and the first PC in fully adjusted models. Barnesiella is
involved in carbohydrate fermentation, competitive inhibition of pathogenic bacteria, and
immunoregulation35-37; it has been shown to be enriched in arthritis-resistant mice38 and associated
with lower levels of active colitis in IL-10−/− mice.39
A critical challenge for observational studies of gut microbiota is the potential for confounding,
particularly as we continue to learn about variables that may influence the gut microbiota.40 In
CARDIA, we were able to account for a large set of sociodemographic, behavioral, and clinical
variables that have been associated with cognitive function. Significant associations between specific
genera and cognitive measures were appreciably attenuated upon adjustment for education,
behavioral confounders, and clinical measures. Furthermore, the confounding structure may vary
across genera; for example, controlling for education attenuated several genus-cognition
associations that were robust to adjustment for other sociodemographic variables or health
behaviors; however, other genus-cognition associations were more sensitive to adjustment for
health behaviors, as compared with education. These results highlight the importance of adjusting
for multiple factors known to be associated with the gut microbiome and the outcome.
Strengths and Limitations

Our study had several strengths. A population-based, sociodemographically diverse cohort, CARDIA
is more representative than patient samples, particularly for the study of cognition among middle-
aged adults. The battery of cognitive assessments assessed multiple domain-specific and global
measures of cognition. Standardized protocols were used for all data collection and quality control,
including participant surveys; clinic-based assessments; and stool collection, processing, and
sequencing.
This study also had several limitations. Our sample size, although larger than many human
microbiome studies, is relatively small for epidemiologic analysis of multiple comparisons,
particularly for assessment of potential effect measure modification by sex and race. We derived gut
microbiota measures based on a single stool sample; however, studies of US populations have
documented relative stability over 6 to 12 months, with between-person differences consistently and

significantly exceeding within-person changes.41,42 The cross-sectional design prevents the

assessment of temporality, and it is possible that declining health itself influences the gut microbial
community. Although we had access to rich covariate data, residual confounding is possible,
particularly for self-reported health behaviors that may have greater measurement error. It is unlikely
that significant cognitive decline has occurred in our middle-aged sample, and results may not
translate to mild cognitive impairment or later disease states. Our analysis relied on 16S rRNA
sequence data, which allowed us to characterize the microbial community with respect to
composition but not directly to function. Future work is needed using whole-metagenomics
sequencing, which would enable direct derivation of gene families and metabolic pathways, as well
as refined taxonomic assignments to the species or strain level.
Conclusions
These data support an association between the gut microbiome and cognitive function. Multivariate
analysis illustrated significant differences in microbial community composition according to
cognition. Taxa-specific analysis was sensitive to covariate adjustment but revealed patterns and
genera-specific associations consistent with mechanistic literature. Specifically, genera within class
Clostridia and genus Akkermansia have been hypothesized to play a role in mechanisms involving gut
membrane integrity and systemic inflammation. These results need to be replicated in larger human
samples. Further investigation, validation, and replication are needed to delineate genomic and
metabolic profiles of gut microbial signals using whole-metagenomics (shotgun) sequencing.
Longitudinal data are required to confirm that gut microbial changes precede relevant physiologic
alterations. The gut microbiota is potentially modifiable through health behaviors and targeted
treatments. Increments in evidence may lead to new opportunities to reduce cognitive decline in
later age through designs of interventions, identification of biomarkers of risk stratification, or
modification of chronic diseases that lead to cognitive decline.
ARTICLE INFORMATION
Accepted for Publication: November 22, 2021.
Published: February 8, 2022. doi:10.1001/jamanetworkopen.2021.43941
Open Access: This is an open access article distributed under the terms of the CC-BY License. © 2022 Meyer K
et al. JAMA Network Open.
Corresponding Author: Katie Meyer, ScD, Nutrition Research Institute, University of North Carolina at Chapel Hill,
500 Laureate Way, Ste 3201, Kannapolis, NC 28081 (ktmeyer@unc.edu).
Author Affiliations: Nutrition Research Institute, University of North Carolina at Chapel Hill, Kannapolis (Meyer,
Lulla); Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill (Meyer); Intramural
Research Program, National Institute on Aging, Bethesda, Maryland (Debroy, Meirelles, Launer); School of
Medicine, Division of Preventive Medicine, University of Alabama at Birmingham (Shikany); Departments of
Psychiatry, Neurology and Epidemiology, University of California, San Francisco (Yaffe).
Author Contributions: Drs Meyer and Lulla had full access to all of the data in the study and takes responsibility for
the integrity of the data and the accuracy of the data analysis.
Concept and design: Meyer, Yaffe, Launer.
Acquisition, analysis, or interpretation of data: Meyer, Lulla, Debroy, Shikany, Yaffe, Meirelles.
Drafting of the manuscript: Meyer, Lulla, Debroy, Yaffe.
Critical revision of the manuscript for important intellectual content: Meyer, Shikany, Yaffe, Meirelles, Launer.
Statistical analysis: Meyer, Lulla, Debroy, Meirelles.
Obtained funding: Meyer, Yaffe, Launer.
Administrative, technical, or material support: Meyer.
Supervision: Meyer.

Conflict of Interest Disclosures: None reported.

Funding/Support: The Coronary Artery Risk Development in Young Adults Study (CARDIA) is supported by
contract Nos. HHSN26820180003I, HHSN26820180004I, HHSN26820180005I, HHSN26820180006I, and
HHSN26820180007I from the National Heart, Lung, and Blood Institute (NHLBI), the Intramural Research
Program of the National Institute on Aging (NIA), and an intra-agency agreement between NIA and NHLBI (No.
AG0005). Additional support for this work was provided by NHLBI grant No. K01-HL127159 and the University of
North Carolina Nutrition Research Institute. CARDIA NHLBI contract funding supported the design and conduct of
the CARDIA examinations, and the collection and management of CARDIA core examination data.
Role of the Funder/Sponsor: Investigator-initiated funding from the NHLBI and NIA, and funding from the NRI,
supported the microbiome study, including design and conduct; sample and data collection, processing, and
management; statistical analysis and interpretation of the data; and manuscript preparation and review. Funders
were not involved in analysis or submission decisions.
Additional Contributions: This manuscript has been reviewed by CARDIA for scientific content.
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SUPPLEMENT.
eTable 1. CARDIA Participant Characteristics for Individuals Who Completed Cognitive Assessments at Year 30
(2015-16), According to Enrollment in the Microbiome Study
eTable 2. Descriptive Statistics for the Seven Cognitive Function Measures
eTable 3. Correlation Coefficients for the Seven Cognitive Function Measures
eTable 4. Descriptive Statistics for Genera
eTable 5. Means (SDs) of Seven Cognitive Function Measures According to Quartiles of the First Two PCoA Axes
(MDS1 and MDS2)
eTable 6. Correlations Between Each of the First Two Axes of the PCoA (MDS1 and MDS2) with Genera Used in the
Analysis
eTable 7. Multivariable-adjusted Associations Between Gut Microbial Alpha and Beta Diversity and Cognition
Measures in CARDIA
eTable 8. PERMANOVA Tests (P Values) for Interaction Between Beta-diversity (PCoA) and Sex (Panel A) and Race
(Panel B)
eTable 9. PERMANOVA Test (P Values) for Sex- (Panel A & B) and Race- (Panel C & D) Stratified Results
eTable 10. P Values for Interaction Between Sex (Panel A) and Race (Panel B) With Alpha Diversity Measures
(Shannon and Richness)
eTable 11. Beta-coefficients (95% CI) and P Values for Alpha-diversity Measures (Shannon and Richness) Within
Strata of Sex (Panel A) and Race (Panel B)
eTable 12. Multivariable-adjusted Regression Coefficients, P Values, Race Interaction P Values, False Discovery
Rate (FDR)-adjusted P Values for Genera With Respect to DSST
Rate (FDR)-adjusted P Values for Genera With Respect to Stroop
Rate (FDR)-adjusted P Values for Genera With Respect to RAVLT
Rate (FDR)-adjusted P Values for Genera With Respect to Category Fluency
Rate (FDR)-adjusted P Values for Genera With Respect to Letter Fluency
eTable 17. Multivariable-adjusted Regression Coefficients, P Values, Race Interaction P Values, False Discovery Rate
(FDR)-adjusted P Values for Genera With Respect to MOCA
Rate (FDR)-adjusted P Values for Genera With Respect to DSST
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to DSST
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to Stroop
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to RAVLT
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to Category Fluency
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to Letter Fluency
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to MOCA
Rate (FDR)-adjusted P Values for Race-Genera Interactions With Respect to PC (1st)
eTable 26. Multivariable-adjusted Regression Coefficients, P Values, and False Discovery Rate (FDR)-adjusted P
Values for Genera Among Black Participants With Respect to DSST

Values for Genera Among Black Participants With Respect to Stroop
Values for Genera Among Black Participants With Respect to RAVLT
Values for Genera Among Black Participants With Respect To Category Fluency
Values for Genera Among Black Participants With Respect to Letter Fluency
Values for Genera Among Black Participants With Respect to MOCA
Values for Genera Among Black Participants With Respect to PC (1st)
Values for Genera Among White Participants With Respect to DSST
Values for Genera Among White Participants With Respect to Stroop
Values for Genera Among White Participants With Respect to RAVLT
Values for Genera Among White Participants With Respect To Category Fluency
Values for Genera Among White Participants With Respect to Letter Fluency
Values for Genera Among White Participants With Respect to MOCA
Values for Genera Among White Participants With Respect to PC (1st)
eTable 40. Multivariable-adjusted Regression Coefficients, P Values, Sex Interaction P Values, False Discovery Rate
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to DSST
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to Stroop
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to RAVLT
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to Category Fluency
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to Letter Fluency
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to MOCA
(FDR)-adjusted P Values for Sex-Genera Interactions With Respect to PC (1st)
Values for Genera Among Male Participants With Respect to DSST
Values for Genera Among Male Participants With Respect to Stroop
Values for Genera Among Male Participants With Respect to RAVLT
Values for Genera Among Male Participants With Respect to Category Fluency
Values for Genera Among Male Participants With Respect to Letter Fluency
Values for Genera Among Male Participants With Respect to MOCA
Values for Genera Among Male Participants With Respect to PC (1st)
Values for Genera Among Female Participants With Respect to DSST
Values for Genera Among Female Participants With Respect to Stroop
Values for Genera Among Female Participants With Respect to RAVLT

Values for Genera Among Female Participants With Respect to Category Fluency
Values for Genera Among Female Participants With Respect to Letter Fluency
Values for Genera Among Female Participants With Respect to MOCA
Values for Genera Among Female Participants With Respect to PC (1st)
eTable 61. Descriptive Statistics for Phyla
Values for Phyla With Respect to DSST
Values for Phyla With Respect to Stroop
Values for Phyla With Respect to RAVLT
Values for Phyla With Respect to Category Fluency
Values for Phyla With Respect to Letter Fluency
Values for Phyla With Respect to MOCA
Values for Phyla With Respect to PC (1st)
eTable 69. Coefficients and P Values for Associations Between Bacteroidota:Firmicutes Ratio With Measures of
Cognitive Function
eFigure. Beta-diversity (PCoA) Differences for Each Cognitive Function Assessment

Association of The Gut Microbiota With Cognitive Function in Midlife

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Association of The Gut Microbiota With Cognitive Function in Midlife

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Original Investigation | Neurology

Association of the Gut Microbiota With Cognitive Function in Midlife

Abstract Key Points

CONCLUSIONS AND RELEVANCE In this cross-sectional study, microbial community composition,

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Covariates and Confounders

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Overall Microbial Community Composition

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Table. Participant Characteristics for Individuals Who Completed

Characteristic No. (%) (N = 597)a

Abbreviations: BMI, body mass index (calculated as weight in kilograms divided

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Category Letter Positive

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measures of cognition. A multivariate measure of between-person differences in microbial

Strengths and Limitations

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significantly exceeding within-person changes.41,42 The cross-sectional design prevents the

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Conflict of Interest Disclosures: None reported.

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