Microbiome function and neurodevelopment in Black infants: vitamin B₁₂ emerges as a key factor

Figures & data

Table 1. Child cohort demographics and neurodevelopmental outcome metrics.

Demographics	Mean ± standard deviation
Gestational age at birth (weeks)	39.17 ± 1.11
Birthweight (kg)	3.088 ± 0.381
Age at evaluation visit (months old)	7.59 ± 3.65
Sex, % male	50%
Delivery mode, % vaginal delivery	81%
Feeding type, % formula (4 months old)	95%
Feeding type, % formula (9 months old)	95%
Solid foods, % in last 7 days (4 months old)	37%
Solid foods, % in last 7 days (9 months old)	58%
Amount of night sleep, hours (4 months old)	7.88 ± 1.82
Amount of night sleep, hours (9 months old)	8.53 ± 1.58
Amount of day sleep, hours (4 months old)	5.45 ± 3.01
Amount of day sleep, hours (9 months old)	4.58 ± 2.55
Number of night wakenings (4 months old)	1.65 ± 0.81
Number of night wakenings (9 months old)	1.00 ± 0.94
Time to put child to sleep, minutes (4 months old)	21.85 ± 13.43
Time to put child to sleep, minutes (9 months old)	27.58 ± 39.39
Bayley Scales of Infant Development III	Mean ± standard deviation
Cognitive composite score	101.90 ± 19.14
Language composite score	88.00 ± 13.02
Expressive communication scaled score	7.20 ± 2.10
Receptive communication scaled score	8.48 ± 3.34
Motor composite score	95.65 ± 15.19
Fine motor scaled score	9.125 ± 3.88
Gross motor scaled score	10.36 ± 3.46
Infant Behavior Questionnaire Revised	Mean ± standard deviation
Effortful control score	5.51 ± 1.01
Surgency score	5.23 ± 1.12
Negative affect score	3.82 ± 1.27
Infant Characteristics Questionnaire	Mean ± standard deviation
Dull score	7.46 ± 3.58
Fussy/Difficult score	14.67 ± 5.16
Unadaptable score	10.75 ± 4.96
Unpredictable score	5.75 ± 3.33
Cortisol response to Face-to-Face Still-Face paradigm	Mean ± standard deviation
Cortisol reactivity (AUCI)	−2.15 ± 9.32

Figure 1. Partial Pearson correlation of neurodevelopmental outcome metrics for a cohort of Black children living in low socioeconomic and high stress environments (average age 7.6 months old; n = 28), after adjustment for gestational age at birth, birthweight, age at assessment visit and sex. Metrics are ordered by hierarchical clustering via the ward linkage method, and optimal clusters are enclosed in rectangles as chosen by the permutation around medoids (i.e., k-medoids) clustering solution with the largest average silhouette width. Significant correlations are indicated as follows: ***p < .001; **p < .01; *p < .05.

Figure 2. Overall α-diversity and β-diversity of the fecal microbiome (n = 28) in relation to the neurodevelopmental outcome metrics. (a) Two community types were determined to be present in the child (average age 7.6 months old) fecal microbiome dataset through the probabilistic method of dirichlet multinomial mixtures. These community types could be described at the taxonomic levels of family and genus, and top microbial genera driving the differential composition of these community types are indicated. Statistically significant Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways differentiating the two community types from gene-set enrichment analysis of the t-scores produced from linear modeling are also displayed by the absolute value of their normalized enrichment scores (NES) and relative significance. ***p < .0001; **p < .001; *p < 0.01. (b) Overall α-diversity (i.e., richness) and β-diversity (i.e., community type) were significantly associated with the Bayley Scales of Infant Development III cognitive and language composite scores in a sex-dependent manner. Significance (p < .05) was determined through multiple regression after adjusting for gestational age at birth, birthweight, and age at assessment visit. Interaction plots of the standardized variables after modeling demonstrate the sex-dependent relationship of richness and these neurodevelopmental outcomes. Estimated marginal means (emmean) with 95% confidence intervals from the standardized variables after modeling demonstrate the sex-dependent relationship of community type and these neurodevelopmental outcomes.

Table 2. Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways and modules of the fecal microbiome that were most significantly positively and negatively associated with each neurodevelopmental outcome metric.

Developmental skills domain neuro-developmental metric	KEGG pathway and [module]	Normalized enrichment score (p value)
BSID-III cognitive composite score	Secondary bile acid biosynthesis ↑ [Cobalamin biosynthesis] ↑ Biosynthesis of amino acids ↓ [Deoxyribonucleotide biosynthesis] ↓	2.4 (p = 7.5 × 10^−6) 2.0 (p = 0.00023) −1.8 (p = 9.5 × 10^−6) −1.7 (p = 3.4 × 10^−6)
BSID-III language composite score	Lipoarabinomannan (LAM) biosynthesis ↑ [Methylaspartate cycle] ↑ Biofilm formation – Escherichia coli ↓ [None significant] ↓	2.3 (p = 9.7 × 10^−7) 2.1 (p = 0.00047) −2.1 (p = 3.1 × 10^−6) N/A
BSID-III expressive communication scaled score	Lipoarabinomannan (LAM) biosynthesis ↑ [Tryptophan metabolism] ↑ Flagellar assembly ↓ [Phenylacetate degradation] ↓	2.7 (p = 1.7 × 10^−6) 1.8 (p = 0.0070) −2.1 (p = 1.0 × 10^−7) −1.7 (p = 0.0041)
BSID-III receptive communication scaled score	Lipoarabinomannan (LAM) biosynthesis ↑ [Cobalamin biosynthesis] ↑ Biofilm formation – Escherichia coli ↓ [Coenzyme A biosynthesis] ↓	2.3 (p = 1.3 × 10^−6) 2.1 (p = 0.00030) −2.1 (p = 1.9 × 10^−5) −1.8 (p = 0.0028)
BSID-III motor composite score	Porphyrin metabolism ↑ [Cobalamin biosynthesis] ↑ Pathogenic Escherichia coli infection ↓ [Phenylacetate degradation] ↓	2.1 (p = 4.3 × 10^−5) 2.1 (p = 0.00035) −1.9 (p = 0.00045) −1.7 (p = 0.0068)
BSID-III fine motor scaled score	O-Antigen nucleotide sugar biosynthesis ↑ [Cobalamin biosynthesis] ↑ Biosynthesis of amino acids ↓ [Leucine biosynthesis] ↓	2.0 (p = 0.00023) 2.0 (p = 0.00058) −2.1 (p = 1.5 × 10^−8) −1.7 (p = 0.0013)
BSID-III gross motor scaled score	Teichoic acid biosynthesis ↑ [Cobalamin biosynthesis] ↑ Biofilm formation – Escherichia coli ↓ [None significant] ↓	2.1 (p = 0.00036) 2.2 (p = 0.0014) −1.9 (p = 2.0 × 10^−5) N/A
IBQ-R effortful control score	Lipoarabinomannan (LAM) biosynthesis ↑ [Methylaspartate cycle] ↑ Biosynthesis of amino acids ↓ [De novo pyrimidine biosynthesis] ↓	2.0 (p = 0.00061) 1.9 (p = 0.0014) −2.3 (p = 1 × 10^−10) −2.1 (p = 2.7 × 10^−5)
IBQ-R surgency score	Lipoarabinomannan (LAM) biosynthesis ↑ [Methanogenesis] ↑ Biosynthesis of amino acids ↓ [De novo pyrimidine biosynthesis] ↓	2.0 (p = 8.9 × 10^−5) 1.8 (p = 0.0016) −2.1 (p = 3.1 × 10^−8) −2.0 (p = 0.00046)
Cortisol reactivity (AUCI)	Phenylalanine metabolism ↑ [Ethylmalonyl pathway] ↑ None significant ↓ [Cobalamin biosynthesis] ↓	2.0 (p = 0.00013) 1.7 (p = 0.0013) N/A −1.9 (p = 0.0050)
Difficult temperament domain neuro-developmental metric	KEGG pathway and [module]	Normalized enrichment score (p value)
IBQ-R negative affect score	Lipopolysaccharide biosynthesis ↑ [KDO2-lipid A biosynthesis] ↑ Lipoarabinomannan (LAM) biosynthesis ↓ [Purine degradation] ↓	2.2 (p = 2.2 × 10^−5) 2.2 (p = 1.1 × 10^−5) −2.3 (p = 8.9 × 10^−6) −2.0 (p = 0.0012)
ICQ dull score	Biosynthesis of amino acids ↑ [De novo pyrimidine biosynthesis] ↑ Steroid degradation ↓ [Dissimilatory sulfate reduction] ↓	2.0 (p = 1.6 × 10^−7) 2.0 (p = 0.00033) −1.7 (p = 0.00028) −1.8 (p = 0.00036)
ICQ fussy/difficult score	Histidine metabolism ↑ [Coenzyme A biosynthesis] ↑ Lipoarabinomannan (LAM) biosynthesis ↓ [Trans-cinnamate degradation] ↓	2.3 (p = 0.00015) 1.9 (p = 0.0067) −1.9 (p = 2.8 × 10^−5) −1.7 (p = 0.0072)
ICQ unadaptable score	Bacterial chemotaxis ↑ [Lysine biosynthesis] ↑ Benzoate degradation ↓ [Purine degradation] ↓	1.8 (p = 0.0091) 1.9 (p = 0.0012) −2.2 (p = 3.3 × 10^−6) −2.0 (p = 9.9 × 10^−5)
ICQ unpredictable score	Histidine metabolism ↑ [Histidine biosynthesis] ↑ Flagellar assembly ↓ [Denitrification] ↓	2.3 (p = 7.3 × 10^−5) 2.6 (p = 1.1 × 10^−5) −2.2 (p = 5.8 × 10^−6) −1.8 (p = 0.00071)

Figure 3. Differential Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways or modules of the fecal microbiome associated with child (average age 7.6 months old; n = 28) neurodevelopmental outcome metrics by sex after gene-set enrichment analysis of t-scores generated from multiple regression models that adjusted for gestational age at birth, birthweight, and age at assessment visit. Estimated marginal means (emmean) for a representative KEGG orthology from each of these pathways/modules are displayed for each sex, with magnitude indicated by the size of circle and direction indicated by the color (yellow = positive association; purple = negative association). Significance (p < .05; false-positive rate < 1%) is indicated from either the model regression term (red colored circle border) or sex interaction (green colored circle border).

Table 3. Microbial taxa contributing to the fecal microbiome Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways/modules significantly associated with the neurodevelopmental domains.

Vitamin B12 associated KEGG pathway/module	Contributing microbial taxa
Cobalamin biosynthesis	Bacteroides dorei Bacteroides vulgatus Blautia spp. Citrobacter freundii Clostridioides difficile Coprococcus catus Corynebacterium variabile Escherichia coli Eubacterium hallii Faecalibacterium prausnitzii Flavonifractor plautii Klebsiella spp. Lactococcus lactis Roseburia hominis Roseburia intestinalis Ruminococcus bicirculans Streptococcus parasanguinis Veillonella dispar Veillonella parvula
Deoxyribonucleotide biosynthesis/De novo pyrimidine biosynthesis (nrdJ exclusively)	Acidaminococcus intestini Bacteroides stercoris Blautia hansenii Corynebacterium falsenii Lactobacillus ruminis
Deoxyribonucleotide biosynthesis/De novo pyrimidine biosynthesis (nrdAB/nrdEF)	Bifidobacterium spp. Blautia spp. Citrobacter spp. Corynebacterium variabile Enterobacter cloacae complex Escherichia coli Haemophilus parainfluenzae Haemophilus sp. HMSC71H05 Klebsiella spp. Leuconostoc citreum Paeniclostridium sordellii Staphylococcus aureus Staphylococcus epidermidis
Ethylmalonyl pathway	Alistipes shahii Citrobacter freundii Escherichia coli Klebsiella spp. Odoribacter splanchnicus
Methylaspartate cycle	Blautia spp. Citrobacter freundii Escherichia coli Klebsiella spp.
Methanogenesis	Blautia coccoides Blautia producta Eubacterium callanderi Eubacterium limosum
Microbial antigen and pathogenicity associated KEGG pathway/module	Contributing microbial taxa
Lipoarabinomannan (LAM) biosynthesis	Bifidobacterium animalis Corynebacterium falsenii Corynebacterium variabile
Teichoic acid biosynthesis	Blautia coccoides Lactobacillus spp. Lactococcus lactis Lactococcus petauri Leuconostoc citreum Paeniclostridium sordellii Streptococcus spp.
Pathogenic E. coli infection	Escherichia coli
Bacterial chemotaxis	Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae
Biofilm formation E. coli	Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae
KDO2-lipid A biosynthesis	Acidaminococcus intestini Alistipes finegoldii Alistipes shahii Bacteroides spp. Escherichia coli Haemophilus parainfluenzae Klebsiella oxytoca Klebsiella pneumoniae Veillonella atypica
Flagellar assembly	Eubacterium eligens Eubacterium rectale Flavonifractor plautii Klebsiella pneumoniae Roseburia intestinalis
Secondary bile acid biosynthesis (7α-dehydroxylation)	Clostridium scindens Dorea sp. D27
Secondary bile acid biosynthesis (3α-dehydrogenation)	Eggerthella lenta Gordonibacter pamelaeae
dTDP-L-rhamnose biosynthesis	Alistipes shahii Anaerostipes hadrus Bacteroides spp. Bifidobacterium longum Blautia spp. Clostridium sp. CAG-299 Coprococcus catus Corynebacterium falsenii Corynebacterium variabile Eubacterium hallii Klebsiella michiganensis Klebsiella pneumoniae Lactobacillus ruminis Odoribacter splanchnicus
Valine, leucine and isoleucine degradation	Acidaminococcus intestini Anaerostipes hadrus Bacteroides spp. Bifidobacterium animalis Blautia spp. Candida tropicalis Coprococcus eutactus Corynebacterium falsenii Corynebacterium variabile Eggerthella lenta Escherichia coli Gordonibacter pamelaeae Haemophilus parainfluenzae Haemophilus sp. HMSC71H05 Klebsiella oxytoca Klebsiella pneumonia Lawsonella clevelandensis Roseburia spp. Rothia mucilaginosa Streptococcus parasanguinis
Purine, histidine and phenylalanine associated KEGG pathway/module	Contributing microbial taxa
Purine degradation	Blautia spp. Coprococcus catus Corynebacterium variabile Enterobacter cloacae complex Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae
Histidine biosynthesis	Acidaminococcus intestini Alistipes finegoldii Anaerostipes hadrus Bacteroides spp. Bifidobacterium spp. Blautia coccoides Ruminococcus torques Citrobacter freundii Clostridioides difficile Coprococcus catus Corynebacterium falsenii Corynebacterium variabile Enterobacter cloacae complex Escherichia coli Eubacterium eligens Klebsiella spp. Lactobacillus ruminis Lactococcus lactis Odoribacter splanchnicus Parabacteroides distasonis Roseburia intestinalis Streptococcus spp. Veillonella dispar Veillonella parvula
Histidine degradation	Acidaminococcus intestini Alistipes finegoldii Alistipes shahii Bacteroides spp. Bifidobacterium longum Blautia coccoides Blautia producta Citrobacter youngae Clostridium sp. CAG-299 Enterobacter cloacae complex Escherichia coli Klebsiella spp. Streptococcus parasanguinis
Phenylacetate degradation	Citrobacter freundii Citrobacter youngae Enterobacter cloacae complex Escherichia coli Klebsiella spp.
Trans-cinnamate degradation	Escherichia coli Klebsiella spp.

Figure 4. The significant (p < 0.05) estimated marginal linear trends (emtrend) between fecal bile acid content and the Bayley Scales of Infant Development III cognitive composite score by sex in a cohort of Black children living in low socioeconomic and high stress environments (average age 7.6 months old; n = 28), after adjustment for gestational age at birth, birthweight, and age at assessment visit.

Data availability statement

The data that support the findings of this study are openly available in NCBI SRA at http://www.ncbi.nlm.nih.gov/bioproject/895372, reference number PRJNA895372, in addition to the provided supplementary tables.