Methodological recommendations for human microbiota-gut-brain axis research.

Observational studies have determined numerous correlations between sequence-based gut microbiota data and human mental traits. However, these associations are often inconsistent across studies. This inconsistency is one of the reasons that mechanistic validation studies of the observed correlations are lagging, making it difficult to establish causal associations. The absence of consistent study findings may partially be due to the lack of clear guidelines for identifying confounders of relations between complex microbial communities and mental conditions. Gut microbial complexity also impedes deciphering microbiota-host relations by using a single analytical approach. The aim of the current review is to help solve these problems by providing methodological recommendations for future human microbiota-gut-brain axis research on the selection of confounders, the use of integrative biostatistical methods, and the steps needed to translate correlative findings into causal conclusions.

Development of the gut microbiota in the first 14 years of life and its relations to internalizing and externalizing difficulties and social anxiety during puberty.

Relations between the gut microbiota and host mental health have been suggested by a growing number of case-control and cross-sectional studies, while supporting evidence is limited in large community samples followed during an extended period. Therefore, the current preregistered study ( https://osf.io/8ymav , September 7, 2022) described child gut microbiota development in the first 14 years of life and explored its relations to internalizing and externalizing difficulties and social anxiety in puberty, a period of high relevance for the development of mental health problems. Fecal microbiota composition was analysed by 16S ribosomal RNA gene amplicon sequencing in a total of 1003 samples from 193 children. Through a clustering method, four distinct microbial clusters were newly identified in puberty. Most children within three of these clusters remained in the same clusters from the age of 12 to 14 years, suggesting stability in microbial development and transition during this period. These three clusters were compositionally similar to enterotypes (i.e., a robust classification of the gut microbiota based on its composition across different populations) enriched in Bacteroides, Prevotella, and Ruminococcus, respectively. Two Prevotella 9-predominated clusters, including one reported by us earlier in middle childhood and the other one in puberty, were associated with more externalizing behavior at age 14. One Faecalibacterium-depleted pubertal cluster was related to more social anxiety at age 14. This finding was confirmed by a negative cross-sectional relation between Faecalibacterium and social anxiety in the 14-year-olds. The findings of this study continue to map gut microbiota development in a relatively large community sample followed from birth onwards, importantly extending our knowledge to puberty. Results indicate that Prevotella 9 and Faecalibacterium may be relevant microbial taxa in relation to externalizing behavior and social anxiety, respectively. These correlational findings need validations from other similar cohort studies, as well as well-designed mechanistic pre-clinical investigations before inferring cause and effect.

Gut microbiota and child behavior in early puberty: does child sex play a role?

A growing number of studies have indicated relations between the gut microbiota and mental health. However, to date, there is a scarcity of microbiota studies in community samples in early puberty. The current preregistered study (https://osf.io/wu2vt) investigated gut microbiota composition in relation to sex in low-risk children and explored behavioral associations with gut microbiota composition and metabolites in the same samples, together with the potential role of sex. Fecal microbiota composition was analyzed in 12-year-old children (N = 137) by 16S rRNA gene sequencing and quantitative PCR. Modest sex differences were observed in beta diversity. Generalized linear models showed consistent behavioral relations to both relative and absolute abundances of individual taxa, including positive associations between Parasutterella and mother-reported internalizing behavior, and negative associations between Odoribacter and mother-reported externalizing behavior. Additionally, Prevotella 9 was positively related to mother-reported externalizing behavior, confirming earlier findings on the same cohort at 5 years of age. Sex-related differences were found in behavioral relations to Ruminiclostridium 5, Alistipes, Streptococcus, Ruminiclostridium 9, Ruminococcaceae UCG-5, and Dialister, for relative abundances, as well as to Family XIII AD3011 group and an unidentified bacterium within the Tenericutes, for absolute abundances. Limited behavioral relations were observed regarding alpha diversity and fecal metabolites. Our findings describe links between the gut microbiota and child behavior, together with differences between child sexes in these relations, in low-risk early pubertal children. Importantly, this study confirmed earlier findings in this cohort of positive relations between Prevotella 9 and externalizing behavior at age 10 years. Results also show the merit of including absolute abundances in microbiota studies.

A longitudinal study of the gut microbiota during the first three years of life: Links with problem behavior and executive functions at preschool age.

Early life is a sensitive period when microbiota-gut-brain interactions may have important impact on development. This study investigated the associations of the gut microbiota in the first three years of life (two, six, and 12 weeks, and one and three years) with problem behavior and executive functions in N = 64 three-year-old children. Higher relative abundance of Streptococcus at the age of two weeks, as well as its trajectory over time (including ages two, six and 12 weeks, and one and three years), was related to worse executive functions. Higher relative abundance of [Ruminococcus] torques group at the age of three years, as well as its trajectory from one to three years, was associated with less internalizing behavior. Besides, several robust age-specific associations were identified: higher Bifidobacterium relative abundance (age three years) was associated with more internalizing and externalizing issues; higher Blautia relative abundance (age three years) was linked to less internalizing behavior; and increased relative abundance of an unidentified Enterobacteriaceae genus (age two weeks) was related to more externalizing behavior. Our findings provide important longitudinal evidence that early-life gut microbiota may be linked to behavioral and cognitive development in low-risk children.

Can gut microbiota throughout the first 10 years of life predict executive functioning in childhood?

Animal models suggest that the gut microbiota can influence cognitive development and functioning via various pathways. In line with that, a first human study found associations between infant fecal microbiota composition and cognition at 2 years of age. This longitudinal study investigated whether fecal microbiota composition in infancy and childhood is associated with executive functioning in childhood. We followed healthy individuals from birth to their 10th year of life. Executive functioning was assessed using the Digit Span working memory test at 10 years of age and the ecologically valid Behavior Rating Inventory for executive functioning at 8 and 10 years. Stool samples were collected at month 1, 3 and 4 as well as at 6 and 10 years. The V4 region of the 16S ribosomal RNA was analyzed to determine microbial composition at the genus level. Using established statistical techniques for microbiota analysis, we did not find associations between fecal microbiota composition and executive functioning after accounting for breastfeeding, maternal education, child sex and age. Our study results are most compatible with the absence or only a weak relationship between infant and childhood fecal microbiota composition and executive functioning in childhood in healthy community children.

Development of the gut microbiota in healthy children in the first ten years of life: associations with internalizing and externalizing behavior.

BACKGROUND: Increasing evidence indicates that psychopathological disorders are associated with the gut microbiota. However, data are largely lacking from long-term longitudinal birth cohorts, especially those comprising low-risk healthy individuals. Therefore, this study aims to describe gut microbiota development in healthy children from birth till age 10 years, as well as to investigate potential associations with internalizing and externalizing behavior. RESULTS: Fecal microbial composition of participants in an ongoing longitudinal study (N = 193) was analyzed at 1, 3 and 4 months, and 6 and 10 years of age by 16S ribosomal RNA gene sequencing. Based on these data, three clusters were identified in infancy, two of which were predominated by Bifidobacterium. In childhood, four clusters were observed, two of which increased in prevalence with age. One of the childhood clusters, similar to an enterotype, was highly enriched in genus-level taxon Prevotella_9. Breastfeeding had marked associations with microbiota composition up till age 10, implying an extended role in shaping gut microbial ecology. Microbial clusters were not associated with behavior. However, Prevotella_9 in childhood was positively related to mother-reported externalizing behavior at age 10; this was validated in child reports. CONCLUSIONS: This study validated previous findings on Bifidobacterium-enriched and -depleted clusters in infancy. Importantly, it also mapped continued development of gut microbiota in middle childhood. Novel associations between gut microbial composition in the first 10 years of life (especially Prevotella_9), and externalizing behavior at age 10 were found. Replications in other cohorts, as well as follow-up assessments, will help determine the significance of these findings.

Lactobacillus casei Strain Shirota Alleviates Constipation in Adults by Increasing the Pipecolinic Acid Level in the Gut.

The benefits of probiotics for constipation are widely accepted, but the mechanisms involving gut metabolites are unclear. In this study, we investigated the effects of Lactobacillus casei strain Shirota (LcS) on constipated patients and revealed that a metabolite mediator is involved in the LcS-induced constipation alleviation. Sixteen constipated patients and 22 non-constipated participants were recruited. The subjects consumed 100 mL of an LcS beverage (108 CFU/mL) per day for 28 days. The fecal non-volatile metabolites were determined by GC/MS, and the targeted metabolites were further verified in a constipated mouse model. In constipated patients, LcS intervention significantly improved defecation frequency (from 4.81 to 7.81 times per week, p < 0.05), stool consistency (from 2.52 to 3.68, p < 0.05) and constipation-related symptoms. A total of 14 non-volatile fecal metabolites were obtained as potential constipation-related metabolites that were regulated by LcS. Among these metabolites, pipecolinic acid (PIPA) had a significant positive correlation with defecation frequency in constipated patients. PIPA significantly promoted the small intestinal propulsive rate (from 25.45 to 39.68%) and increased the number of fecal pellets (from 30.38 to 57.38 pellets) in constipated mice (p < 0.05). The 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) in colonic tissue may be partly involved in PIPA-mediated constipation alleviation. In conclusion, PIPA was a metabolic mediator in the gut that participated in LcS-induced constipation alleviation.

Differential Effects of Lactobacillus casei Strain Shirota on Patients With Constipation Regarding Stool Consistency in China.

BACKGROUND/AIMS: Probiotics are expected to confer benefits on patients with constipation, but how probiotics act on constipated patients with variable stool consistencies remains unclear. We investigated the effect of Lactobacillus casei strain Shirota (LcS) on constipation-related symptoms, especially stool consistency, of constipated patients. METHODS: Constipated patients meeting the Rome III criteria were divided into 3 groups according to the Bristol Stool Form Scale (BSFS): hard (hard stool [HS], BSFS ＜ 3), normal (normal stool [NS], ≤ 3 BSFS ≤ 4), and soft (soft stool [SS], 4 ＜ BSFS ≤ 5) stools. Subjects in each group consumed a probiotic beverage containing 10¹° colony-forming units of LcS daily for 28 days. RESULTS: LcS intervention significantly alleviated constipation-related symptoms and increased defecation frequency in all subjects. Four weeks of LcS supplementation softened the hard stools in HS, hardened the soft stools in SS, and did not alter the ideal stool consistency in NS. The short-chain fatty acid (SCFA) concentrations were highest in SS, followed by NS and HS. LcS intervention increased the stool SCFA levels in HS but reduced or did not alter the levels in NS and SS. LcS intervention increased the Pseudobutyrivibrio and Roseburia abundances in HS and decreased the Pseudobutyrivibrio abundance in SS. CONCLUSIONS: LcS supplementation improved the constipation-related symptoms in constipated subjects. Differences in baseline stool consistency could result in different anti-constipation effects of LcS intervention. LcS balanced the stool consistency-softened the HS and hardened the SS. These effects could be associated with modulation of the gut microbiota and SCFA production.