In the present study, we established two IBS rat models using maternal separation (MS) and multiple early adversities (MAM) modeling and investigated their intestinal microbiota. Rats from both IBS models exhibited visceral hypersensitivity compared with the control group rats. However, the two model groups exhibited differences in the extent of visceral pain sensitivity and fecal water content. The microbial community structure from rats with the two visceral hypersensitivity models exhibited significant differences compared to the controls, while communities from the two model groups also exhibited significant differences between them. Furthermore, community functional predictions suggested that rats from the two models exhibited different abundances of metabolisms and pathways associated with structural changes. Several common and distinct characteristic differences were also observed between rats from the two model groups. Some of these bacterial taxa may play a mediating role in the association between early adversity and specific pathological changes of IBS.
Several studies have evaluated early adversity events and alterations in intestinal flora diversity. Some of these have suggested that exposure to early adversity can affect the gut microbiota, manifesting as a reduction in microbial diversity and richness [17, 18]. However, other studies have reported no changes in diversity in these scenarios . Comparison of five alpha-diversity indices between different model groups did not yield any significant differences in this study. The apparent discrepancies in associations between microbial community alpha-diversity and early adversity could be explained by differences in study designs and environments, or even by factors of the same study including the type of early adverse events that were used and the timing of material collection, which can lead to variable results. For example, Zhou et al.  evaluated the composition and diversity of the gut microbiota of MS, PI-IBS, and control rats at three different time points. No differences in alpha diversity were observed among the three groups in the third week and the eighth week, only the MS group exhibited a significant decrease in the Simpson and Shannon indices at week 12, while the PI-IBS group did not exhibit significant differences.
Inter-individual variation within host cohorts can also reflect the stability of gut microbiota. Healthy and homeostatic gut microbiota share higher similarities in the composition and richness of microbial taxa, while those with imbalanced microbiota exhibit drastic changes and tend to have higher inter-individual variation. Inter-group distances in the two model cohorts of this study exhibited significantly higher variation than those of the control group (Fig. 3c). Thus, induced early negative events resulted in a significant increase in the instability of gut flora. This result supports the hypothesis that exposure to early adversity can induce significant dysbiosis of fecal microbiota . The PCoA analyses based on the unweighted UniFrac distance metric indicated that the microbiota from the three model groups could be completely divided into three disparate clusters. Further comparison of intra-group distances indicated that the distances between the two model groups were significantly higher than the distances between those groups and control rat communities. These results indicated that the flora structure of the two visceral hypersensitive model rats significantly changed relative to the controls, and that obvious differences were present between the two model groups.
To further evaluate the effects of different modeling processes on gut floral compositions, the composition and functionality differences of the communities among the model and control groups were further investigated. The two model groups exhibited several significantly altered taxonomic group abundances compared to the control group. The bacterial taxonomic differences unique to the MAM group comprised five taxa, where the genus Butyricimonas decreased significantly and Vampirovibrio along with the family, order, class, and the phylum to which it belongs all increased significantly compared to the control group. Bacterial taxonomic differences unique to the MS group involved 20 taxa, where Corynebacterium and the family to which it belongs increased significantly, while many taxa decreased significantly compared to the control group including Rothia and the family to which it belongs; Elusimicrobium and the family, order, class, and phylum to which it belongs; Romboutsia and the family to which it belongs; Clostridium IV; Allobaculum and the family, order, and class to which it belongs; in addition to Parasutterella and the family, order, and class to which it belongs.
Some studies have suggested an association and role for Butyricimonas and Vampirovibrio with IBS, consistent with our results [21, 22]. Butyricimonas have been reported to produce butyrate that reduces inflammation and helps maintain healthy gut functioning. Therefore, decreased abundances of Butyricimonas in gut systems may play an inflammation-based role in the pathogenesis of IBS . Vampirovibrio has been reported as significantly more abundant in trinitrobenzene sulfonic acid-induced PI-IBS rat model individuals, and its lowered abundance has been associated with the alleviation of symptoms for visceral hypersensitivity after treatment .
The abundances of Parasutterella species and their related taxonomic groups were significantly lower in the MS group compared to the control group. These results contrast with those of Chen et al. . The latter group found that Parasutterella was abundant in the stool of IBS patients. However, our results are consistent with other studies indicating that Parasutterella abundances are associated with beneficial outcomes. Zhang et al.  reported that Parasutterella abundances were significantly lower in CDI patients and asymptomatic carriers than in healthy controls, while Kreutzer et al.  also reported a negative correlation between Parasutterella abundances and high-fat diet-induced hypothalamic inflammation. Although the association of Parasutterella and different health outcomes is clearly controversial, these observations nevertheless indicate that Parasutterella has a role in modulating the microbial activities and host responses in certain disease states and, thus, necessitate further investigation.
The associations identified here between Corynebacterium, Rothia, Elusimicrobium, Romboutsia, Clostridium IV, and Allobaculum with maternal separation stress have not been reported elsewhere. Consequently, their associations with IBS require additional confirmation in further studies.
Predictive analysis of microbiota functionalities also suggested that changes in the microbial composition of the two model groups corresponded to functional differences associated with diverse metabolisms and pathways. Specifically, we observed higher abundances of the pathways in the MS group compared to the control group involving the citrate (TCA) cycle (ko00020), protein export (ko03060), carbohydrate digestion and absorption (ko04973), and NF-kappa B signaling (ko04064), along with lower abundances of alanine, aspartate, and glutamate metabolism (ko00250), non-ribosomal peptide structures (ko01054), tyrosine metabolism (ko00350), and porphyrin and chlorophyll metabolism (ko00860) (Fig. 4e). In the MAM group, pathways that were significantly more abundant than in the control group included epithelial cell signaling in Helicobacter pylori infection (ko05120), galactose metabolism (ko00052), glutathione metabolism (ko004800), and the Fanconi anemia pathway (ko03460), while significantly lower pathways included morphine addiction (ko05032), thiamine metabolism (ko00730), methane metabolism (ko00680), and bacterial chemotaxis (ko02030) (Fig. 4f). These results together support the hypothesis that the complex metabolic interactions between microbes and host tissues are critical to the development of IBS symptoms . Furthermore, they suggest that different modeling strategies may result in IBS symptoms by inducing changes in bacterial flora via different metabolic pathways.
In the present study, the microbial composition of MS group gut communities was associated with the enhanced functional capacity for the NF-kappa B signaling pathway. This result is consistent with previous studies indicating that neonatal MS induces VH and visceral pain in rats that is mediated by activation of TLR4 and the NF-κB signaling pathway [27, 28] and further suggests that changes in the gut microbiomes caused by MS are involved in NF-κB activation in a rat model of IBS.
The microbial composition of the MAM group was also associated with functions related to morphine addiction. Several opioid, cannabinoid, and 5-HT receptors play critical roles in visceral hypersensitivity and abnormal gut motility associated with IBS and also contribute to the maladaptive effects of chronic morphine analgesia including hyperalgesia, tolerance, and dependence [29–31]. Therefore, we speculate that the visceral hyperalgesia induced by MAM modeling may be mediated by interactions between microorganisms and their host receptors. Further investigations incorporating metagenomic analyses at the gene level could help us understand how gut microorganisms affect visceral pain sensation in IBS.
Despite the significant differences in microbial composition and functionalities among the two model groups, six taxa were common in both the model groups relative to the control group communities. Specifically, Alloprevotella were more abundant in both the MS and MAM groups, while Butyricicoccus, Turicibacter, Ruminococcus, and Clostridium sensu stricto along with the family it belongs to were less abundant in both the MS and MAM groups relative to controls. These results were consistent with those from previous studies. For example, Alloprevotella were significantly more abundant in model rats with early adversity stimuli compared to controls in one study . In addition, Turicibacter were significantly less abundant in IBS-D patients . Ruminococcus, Butyricicoccus, and Clostridium sensu stricto are all short-chain fatty acid-producing genera that are considered beneficial for gastrointestinal tract functioning [33–35]. Furthermore, Ruminococcus spp. were less abundant in IBS patients relative to controls in another study . Moreover, several studies have shown that the abundances of Butyricicoccus were lower in ulcerative colitis patients and patients with inflammatory disease in general [35, 37, 38]. These microbial signatures exhibited similar abundance patterns in both early adversity modeling procedures, indicating that the responsive species may be involved in common characteristics of the complex pathophysiological changes that cause IBS. Thus, the variation in these taxa could provide clues to the etiology of IBS and potentially lead to novel therapies.
The changes in the gut microbiota and pathophysiological indicators described in this study were both induced by early adversity events. To further assess the contribution of altered gut microbial compositions to the association between early adversity and altered physiological outcomes, mediation analysis was performed to investigate the potential mediating effects of microbial populations on pathophysiological changes induced by early adversity events. Butyricimonas and Butyricicoccus populations were predicted to partially mediate MAM exposure-induced visceral hypersensitivity, while Corynebacterium was predicted to partially mediate MS exposure-induced visceral hypersensitivity. Thus, these genera might play critical roles in the development of early adversity-induced visceral hypersensitivity and may also represent targets for understanding the mechanisms underlying microbe-mediated visceral hypersensitivity.
A limitation of the present study is the lack of proven causal relationships between alterations in microbiota characteristics and visceral pain behaviors, despite the abundance of evidence suggesting that microbial compositions play roles in the development of visceral hypersensitivity. Future studies could deconvolute these effects by focusing on manipulating the gut microbiota through prebiotics, probiotics, or synbiotics to reverse the deleterious effects of early adversarial events on visceral pain or other IBS-related behaviors.