This exploratory study aimed to identify possible correlations between gut microbial community structure and brain architecture. The main findings of the study were (1) The identification of IBS subgroups based on distinct microbial clusters with only one of these subgroups (IBS1) showing gut microbial and some behavioral and clinical differences; (2) The identification of differences in the abundance of certain microbial taxa between IBS1 and HCs; and (3) The correlation of microbial taxa and imputed metagenes with structural brain alterations primarily in sensory integration and salience network regions in IBS1. To our knowledge, this is the first report demonstrating a correlation between the architectures of the brain and the gut microbiota in a distinct subgroup of IBS patients. These findings suggest a possible influence of the gut microbiota and their metabolites on specific brain structures which may play a role in the pathophysiology of altered sensory processing in IBS.

Identification of IBS subgroups, based on microbiome diversity and relative abundance of the phyla Firmicutes and Bacteroidetes

Both hierarchical clustering using average linkage and PCoA analysis on unweighted Unifrac distances indicated that microbial signatures can be used to identify two subgroups among the IBS subjects, one that has a microbial composition similar to HCs, and one that shows a distinct gut microbial signature. This differentiation was also supported by our Random Forest classifier. Identification of the subgroups was not associated with sequencing depth. As a group, all IBS subjects showed significantly greater alpha diversity and richness than HCs, a difference that was largely explained by the greater diversity within the IBS1 subgroup. Higher alpha diversity has previously been reported in an IBS subgroup [10], in patients with celiac disease [51], and with autism spectrum disorder [52], all syndromes which are often accompanied by IBS-like symptoms. Even though an increased microbial diversity has been associated with diets high in fruits, vegetables, and fiber [53], a detailed dietary analysis between the groups did not reveal significant differences in dietary intake.

Subjects within the IBS1, but not the HC-like group, differed from the HC group in the relative abundances of the phyla Firmicutes and Bacteroidetes (F-B ratio). While the abundance of Firmicutes was significantly greater in IBS1, that of Bacteroidetes was lower. In contrast, the HC-like IBS group did not differ from the HC with respect to these relative abundances. The finding of gut microbiota-based IBS subgroups and some of their gut microbial composition is similar to findings recently reported by Jeffery et al. [10], even though they identified two IBS clusters that differed from HCs in addition to a subgroup similar to HC. The finding of IBS subgroups based on gut microbial composition that had identical clinical symptoms suggests that the difference in microbial architecture is either not necessary for symptom generation in IBS, or that identical clinical presentations can be caused by different underlying mechanisms, one involving gut microbial alterations. Similar to the current finding in the IBS1 subgroup, both clusters in Jeffrey’s study showed a greater abundance of Firmicutes and a lower abundance of Bacteroidetes. Altered F-B ratios have been reported in other chronic diseases [54], with increased F-B ratios reported in both preclinical and clinical studies of obesity, metabolic syndrome, and high fat intake [55,56,57].

The reason for the increased F-B ratio found in this and in previous studies in IBS subjects [8, 9] remains unknown. F-B ratios in healthy individuals are highly variable, despite similarities in microbial function [58]. However, age [59] and diet [55, 57, 60] are important modulatory factors. For example, animal models of obesity, obese individuals, those with metabolic syndrome, and those on a Western diet with high animal fat content were found to have increased F-B ratios [55,56,57]. Fatty acids in high fat diets, such as in the typical North American diet, have been shown to increase the F-B ratio, and this increase has been associated with an increase in gut epithelial permeability and low grade inflammation [61]. It is intriguing to speculate that the increased F-B ratio in a subgroup of IBS patients is related to alterations in epithelial permeability and low grade inflammation which have been implicated as possible disease mechanisms in IBS [62]. Even though we observed trends for higher BMI and higher plant-derived fat intake in the IBS1 group, these differences did not reach statistical significance. Specific dietary habits, such as increased consumption of fat, have not been reported for IBS [63, 64]. No other dietary differences between the groups were identified.

In addition to the differences in relative abundances at the phylum level, several differences at lower taxonomic levels were also observed. The IBS1 group showed a greater relative abundance of several Firmicutes-related taxa, including members of Bacilli and Clostridia at the class level, of Bacilli-associated Lactobacillales at the order level, and of Holdemania at the genus level. The IBS-associated enrichment in Clostridia is particularly interesting in light of the finding that select spore-forming bacteria, dominated by Clostridia Cluster IV and XIVa, sufficiently induce serotonin biosynthesis by colonic enterochromaffin cells [65]. This aligns well with the reported links between intestinal serotonin dysregulation and IBS [66]. Holdemania are commonly found in the healthy gut, but there are few generalizable results for this genus. IBS1 differed in these relative abundances from both HC-like IBS and HCs, with the exception of differences in the abundance of Lactobacillales, which were only seen in comparison to HCs. This finding is surprising in view of the common recommendation of probiotics to treat IBS symptoms, the majority of which contain Lactobacilli. As acute laboratory stressors have been shown to decrease Lactobacilli in the stool in both clinical and preclinical studies, one would expect a reduction of Lactobacilli in a stress-sensitive disorder like IBS.

In addition to the increased abundance in some microbes, the IBS1 group had lower relative abundances of Bacteriodia at the class level, of Bacteroidales at the order level, and of Parabacteriodes at the genus level. All these differences were significant when compared to the HC group, but not the HC-like IBS group. The random forest analysis showed that OTUs contributing to the differentiation of IBS1 from HC gut communities included members of the genera Blautia, Streptococcus, Faecalibacterium, and Bacteroides.

The reasons that the group differences in relative abundance at lower taxonomic levels differed from those reported by others [9, 10, 67] are unknown, but may include differences in patient populations, diet, DNA extraction techniques, and the primers used for amplicon generation, as well as differences with respect to bioinformatic pipelines, data transformations, and the statistical approaches applied to the data.

Correlation of gut microbial composition with behavioral and clinical parameters

Similar to Jeffrey’s findings, we found few correlations between the gut microbial-based subgroups and clinical parameters such as IBS symptom severity, predominant bowel habit, or medication use, except for a moderate correlation with symptom duration. As subjective ratings of predominant bowel habits generally show poor correlations with colonic transit times, and the colonic transit times are normal in the majority of IBS patients regardless of reported predominant bowel habit, the observed lack of correlation of such ratings with microbial composition is not surprising. As commonly observed in clinical studies, IBS patients as a group had a significantly higher level of anxiety symptoms while IBS1 and HC-like IBS did not differ from each other. However, no correlations between anxiety (or depression) symptom scores and microbial parameters were observed.

Interestingly, IBS1 had significantly greater scores on the emotional scale of the ETI than both the HC-like IBS and HCs. While the relative abundance of the Firmicutes-associated class Bacilli was positively correlated with ETI total score, as well as with scores in the sexual and emotional subscales of the ETI, no correlations with EAL were found for the Firmicutes-associated class Clostridia, or the Bacteriodetes-associated class Bacteroidia. Even though the observed correlation between a history of ELAs and the microbiome needs to be confirmed in future studies, one may speculate that brain driven disturbances of the gut microbial environment in early life [12] may have a long lasting effect on gut microbial composition persisting throughout life, which in turn may lead to further changes in brain structure/function.

Correlation with brain structures

Moderate-sized correlations with brain structure were observed for certain Firmicutes- and Bacteroides-associated taxa. For example, the Firmicutes-associated Clostridia (higher in IBS1) and the Bacteroidetes-associated Bacteroidia (lower in IBS1) showed correlations with the volume of several subcortical brain regions involved in sensory integration and modulation and the motor cortex. For the majority of these regions, increased volumes were observed with decreases in Bacteroidia taxa and increases in the Clostridia taxa characterizing IBS1. On the other, decreased volumes of the anterior insula and ventral prefrontal regions were associated with the taxa profile of IBS1. Whether Clostridia-mediated modulation of peripheral serotonin levels [65] may be involved is unclear, increasing evidence reveals microbiome-mediated changes in neurochemical signaling and neurophysiology. The abundance of the Firmicutes-associated Bacilli (increased in IBS1) was related to only a few regional brain volumes including positive correlations with the right nucleus accumbens and subregions of the frontal gyrus, and negative correlations with other prefrontal cortices and the posterior cingulate cortex.

Previous structural and white matter studies have shown IBS-related alterations in some of the same regions, with IBS patients showing larger gray matter volumes [68] and altered white matter tracts in the thalamus and basal ganglia [69], and reduced gray matter volumes in insula and prefrontal cortices [68]. Several possible explanations for these structural brain changes have been proposed, including genetic and epigenetic factors, the effect of gut microbial metabolites, as well as the effect of longstanding increased sensory signaling from the periphery [6].

The biological mechanisms underlying the observed correlations remain to be determined. For the sensory brain regions, it is conceivable that neuroactive or proinflammatory metabolites generated by altered gut microbiota reach the brain, inducing neuroplastic changes. As most patients with IBS symptoms have a longstanding history of symptoms, often dating back to childhood, it is likely that such altered gut microbiota to brain signaling could have shaped the brain from early on in life. This view is also consistent with the observed correlation of microbial composition with EALs.

In support of this possibility, we found that the surface area of the posterior insula was associated with the predicted abundance of 20 bacterial genes increased in the IBS1 group. The posterior insula is considered the primary visceral cortex and was chosen a priori without corrections for type I error. The identified genes included two that influence synthesis/degradation of GHB and glutamate. GHB is a neurotransmitter found naturally at high levels in the intestine that inhibits intestinal peristalsis via GABA B receptors and has sedative effects in the CNS [70, 71]. Glutamate is an excitatory neurotransmitter in the enteric nervous system and in the brain where it also plays an important role in synaptic plasticity [72]. The posterior insula was also associated with abundance of a subunit of butyryl-CoA:acetate CoA-transferase, an enzyme used by intestinal bacteria such as Faecalibacterium in the final step of butyrate synthesis [73, 74]. Butyrate has histone deacetylase activity and signals through GRP43 and GRP109a (expressed by enterochromaffin cells, vagal afferents, and microglia), all potential mechanisms by which it could influence brain function or structure [75]. Interestingly, a positive regulator of proprionate catabolism operon was also associated with this region of the posterior insular, suggesting a shift in the short-chain fatty acid profile [76]. Association of these metagenes with IBS1 group was largely attributable to a single unclassified member of the Peptostreptococcaceae. It is unclear why this taxon bloomed in a subset of IBS patients, though diet or fiber supplementation are possibilities [77]. Mechanistic studies in rodent models are warranted to investigate these hypotheses.

There are several limitations to this study. The sample was relatively small and composed of both male and female subjects and did not include measures of intestinal transit times. Self-ratings of bowel habits are known to have a poor correlation with intestinal transit time. All correlations with brain structure were cross sectional, and no conclusion about causality can be made from our results. The observed gut microbial changes could be secondary to altered autonomic nervous system output to the gut, changing the microbial environment [78]. Alternatively, the brain changes could be the consequence of altered signaling to the brain through microbial metabolites, or both mechanisms may be involved [6]. Sequencing depth of the microbiome may be a limitation as rare members of the microbiota may be undetected, and differences in sequencing depth may affect relative distributions of microbial taxa. Finally, predicted metagenomics analysis to impute potential metabolites involved in the observed structural brain differences is limited, and results need to be confirmed by metabolomics analyses.