Modulation of visceral sensitivity by faecal microbiota transplantation (FMT): the active role of gut microbiota in the persistence of abdominal pain

Background. Recent ndings linked gastrointestinal disorders characterized by abdominal pain to gut microbiota composition. The present work aimed to evaluate the power of gut microbiota as a visceral pain modulator and, consequently, the relevance of its manipulation as a therapeutic option in reversing the persistence of visceral hypersensitivity consequent to colitis induced by the intra-rectal injection of 2,4-dinitrobezenesulfonic acid (DNBS) in rats. Results: The effect of faecal microbiota transfer (FMT) from viscerally hypersensitive DNBS and naïve donors was evaluated in control rats after an antibiotic-mediated microbiota depletion. FMT from DNBS donors induced a long-lasting visceral hypersensitivity in control rats. Pain threshold trend correlated with major modications in the composition and structure of the gut microbiota at phylum (Proteobacteria and Firmicutes to Bacteroides ratio) and family levels (Enterobacteriaceae, Akkermansiaceae and Lachnospiraceae). Acetic acid was signicantly increased in the recipients FMT from DNBS donors. Gut cytokine prole, as well as tryptophan metabolism were similarly altered after FMT from both DNBS and naïve donors. By contrast, no signicant alterations of colon histology, permeability and monoamines levels were detected. Finally, following FMT from healthy donors to DNBS-treated animals, a counteraction of persistent visceral pain was achieved. Conclusions: The present results provide novel insights into the relationship between intestinal microbiota and visceral hypersensitivity, highlighting the therapeutic potential of microbiota modulation on persistent abdominal pain.


FMT from DNBS donors transfers visceral hypersensitivity
To limit the effects of the antibiotics on visceral sensitivity, we investigated the minimum treatment duration needed to obtain a su cient microbiota depletion prior to proceed with FMT. The culture-based analyses performed on freshly collected faecal pellets showed no viable bacterial cells after 7 days treatment. On day 7, antibiotics induced visceral hypersensitivity as assessed by abdominal withdrawal response (AWR) to colorectal distension (CRD) (Fig. 1A), whereas the viscero-motor response (VMR) was unaltered (Fig. 1B). Antibiotic-induced hypersensitivity was mild on day 12 and completely disappeared on day 18. Starting on day 7 and following the scheme reported in Fig. 1 FMT was performed using faeces from control (FMT CTR ) or from animals in which a severe visceral pain was induced by the intrarectal injection of DNBS (FMT DNBS ).
FMT DNBS induced, in comparison to FMT CTR , a signi cant increase of both AWR and VMR to CRD after the set I of FMT (days 12-18, Fig. 1A and B, respectively). Hypersensitivity was consolidated after the set II of FMT (days 25-32, Fig. 1), in particular when evaluated as AWR in awake animals (until day 39; Fig. 1A). Three weeks after the interruption of treatments (day 46), the effect of FMT DNBS ended and the animals' visceral sensitivity came back to the value registered in controls (Fig. 1A). FMT CTR did not induced signi cant effects on pain threshold, showing no differences in comparison to the group abx + vehicle.
We observed similar results in preliminary experiments employing a longer antibiotic treatment (24 days), in this case though the effect of microbiota transplantation on visceral sensitivity was less clear due to the persistence of antibiotics-induced gut sensitivity alterations ( Figure S1).

Visceral hypersensitivity is accompanied by alteration of the gut microbiota composition
The taxonomic analysis of faecal suspensions, subsequently used for FMT, revealed several differences between control and DNBS-treated animals ( Figure S3A). For instance, the phylum Verrucomicrobia showed diminishing trend in suspensions from DNBS-treated animals used for the FMT. At lower taxonomic level, the Prevotellaceae family was almost absent in suspensions from DNBS-treated animals, while other families were found to be underrepresented, including Akkermansiaceae and Lactobacillaceae; on the other hand, an expansion of the Bacteroidaceae and Tannerellaceae families was identi ed ( Figure S3A).
Analysis of alpha diversity in samples collected after the I and II set of FMT (day 18 and 46), showed a signi cantly greater microbial diversity in samples from the groups of animals receiving abx + FMT CTR and abx + FMT DNBS compared to others ( Figure S3B). This observation suggested a stable engraftment of gut microbiota following transplantation, whereas an antibiotic-driven dysbiosis was still persisting in animals of the abx + vehicle group. Such differences were even more marked on day 46, where the II set of FMT was clearly associated with a higher microbial diversity in samples from abx + FMT CTR and abx + FMT DNBS compare to controls. The long-lasting effect of the antibiotic and antifungal treatment was still evident in samples from animals belonging to the abx + vehicle group that displayed an overall lower microbial richness compared to other groups ( Figure S3B). On day 32, samples from the abx + FMT DNBS group were characterized by a microbial richness slightly higher, although not signi cant, than samples from abx + FMT CTR . Evaluation of beta-diversity through PCoA using the Bray-Curtis metric ( Fig. 2A) showed that samples from the abx + vehicle group clustered away from samples of corresponding controls (i.e. controls at day 18 (Fig. 2B). A similar behaviour was observed in PCoA plots using the weighted UniFrac metric ( Figure S4B). Consistently with the sample clustering observed on beta-diversity PCoA plots, analysis of the taxonomic composition of samples belonging to the abx + FMT CTR and abx + FMT DNBS groups pointed out to several signi cant differences at phylum and family level.
In detail, while no major changes in the relative abundance of most represented phyla were detected at day 18, an expansion of the Lactobacillaceae and Staphylococcaceae families was observed in samples from the abx + FMT DNBS and from the abx + FMT CTR groups (Kruskal-Wallis p < 0.043), respectively ( Fig. 2C). At day 32, marked changes were identi ed in samples from the abx + FMT DNBS at a phylum level, showing a higher Firmicutes to Bacteroidetes ratio and a contraction of Proteobacteria and Actinobacteria (Kruskal-Wallis p = 0.02); at family level, the most noticeable change consisted in a large expansion of Lachnospiraceae and a concomitant contraction of Enterobacteriaceae and Burkholderiaceae in samples from the abx + FMT DNBS (Kruskal-Wallis p = 0.02). Additional statistically signi cant variations (Kruskal-Wallis p < 0.047) have been observed in samples from the abx + FMT DNBS and from the abx + FMT CTR groups at day 32, although occurring at a lower extent ( Fig. 2B and C). At day 46, some of the previously observed signatures were still present in samples from the abx + FMT DNBS group, as the higher Firmicutes to Bacteroidetes ratio, an even more pronounced expansion of the Lachnospiraceae family and a contraction of Enterobacteriaceae and Burkholderiaceae relative abundances (Kruskal-Wallis p < 0.043) ( Fig. 2B and C). Interestingly, a peculiar variation trend was observed in the relative abundance of Akkermansiaceae, being overall equally represented in samples from the abx + FMT CTR and abx + FMT DNBS groups at Day 18 and 46 and almost disappeared in samples from the abx + FMT DNBS group at Day 32 (Fig. 2C).
Changes in the levels of faecal SCFAs are associated with visceral hypersensitivity Faecal pellets were collected from animals undergoing to FMTs 7 days after the I and the II set of treatment (days 18 and 32) and 3 weeks after the last treatment (day 46). An increase of the total amount of SCFAs was found in the abx + FMT DNBS group in respect to the abx + FMT CTR . This difference was detectable already on day 18, becoming signi cant on day 32 and extinguished on day 46 (Fig. 3A). Further in-depth analysis revealed that alteration on day 18 were due to an overall increase of fatty acids, while on day 32 acetic acid alone was found to be raised, while butyric acid signi cantly decreased Correlation analyses between concentration of SCFAs and relative abundances of microbial taxa that were previously found to statistically differ in abx + FMT CTR and abx + FMT DNBS groups revealed that some time-dependant associations were identi able. In detail, after the I set of FMT at day 18, Peptococcaceae and Clostridiales vadin BB60 group were found to be negatively correlated with butyric acid (Spearman r range: -0.857, -0.821; P < 0.035), while the Lactobacillaceae family was positively correlated with acetic acid (Spearman r: +0.850, P: 0.02). After the II set of FMT at day 32, several bacterial families were found to be negatively correlated with acetic acid (Spearman r range: -0.778, -0.881; P < 0.030), including Burkholderiaceae, Enterobacteriaceae, Bi dobacteriaceae, Enterococcaceae, Mollicutes RF39, as well as two phyla including Actinobacteria and Proteobacteria (Spearman r range: -0.928, -0.833; P < 0.015). On day 46, Rikkenellaceae and Anaeroplasmataceae were positively correlated with iso-Butyric acid (Spearman r range: 0.898, 0.803; P < 0.025) FMT including the source does not affect colon histology Colon samples were collected on days 7 (24 h after the end of antibiotic treatment), 32 (when the effect of FMT on pain was well established) and 46 (when the effect of FMT extinguished). The Macroscopic Damage Score (MDS; Fig. 4A) highlights the presence of hyperaemia and a slight thickening of wall after the antibiotic treatment (day 7). These results were con rmed by microscopic analysis: an in ltration of neutrophils in mucosa and sub-mucosa were revealed in the animals receiving the antibiotic treatment (day 7; black arrow; Fig. 4B). The tissue alteration disappeared following to the interruption of antibiotic treatment. No microscopic differences were observed in the animals receiving FMTs (day 32 and 46; Fig. 4B).
FMT does not affect gut permeability factors Gut permeability was indirectly evaluated by assessing the plasma levels of lipopolysaccharide binding protein (LBP; Fig. 5A) and also by measuring the mRNA expression of the tight junctions occludin and Zo-1 in the colon ( Fig. 5B and 5C, respectively).
No signi cant alteration in the plasma levels of LBP was found among the experimental groups, a trend towards increase after the antibiotic treatment was observed on day7 (Fig. 5A). On the other hand, no difference was found in the plasma levels of LBP between CTR and DNBS donors (Fig. 5A).
FMT alters gut cytokines pro le, irrespective of the donor The expression of TNF-α, IL-6, IL-10 and TGF-β mRNA level was measured in the colon by RT-qPCR. The antibiotic regimen caused a long-lasting up regulation of both pro-and anti-in ammatory cytokines expression (day 7; Fig. 6). This deregulatory effect of antibiotics did not change was not xed by FMT (day 32; Fig. 6).
On the other hand, the cytokines pro le of the FMT recipients did not match the pattern observed in the donors. The expression of both IL-6 and TGF-β resulted augmented in DNBS donors in respect to CTR donors but both FMT DNBS and FMT CTR recipient animals showed overexpression (day 32, Fig. 6B and 6D, respectively). Further, TNF-alpha and IL-10, unaltered in DNBS donors, were respectively down and upregulated in FMT DNBS recipients (day 32; Fig. 6A and 6B, respectively). On day 46 all cytokines were still upregulated in the abx + vehicle group and in the abx + FMT CTR group. By contrast, in the abx + FMT DNBS all cytokines, with exception of TGF-β, returned to the values observed in controls (Fig. 6).
Transfer of microbiota from healthy controls counteracts post-in ammatory visceral pain persistence Finally, we tested the hypothesis that visceral pain could be suppressed or reduced by FMT from healthy donors. Pain threshold was assessed by evaluating the animal AWR in response to CRD stimulus (Fig. 7). As expected, 7 days after the induction of the damage, the abdominal withdrawal response to CRD was signi cantly higher in both the groups treated with DNBS. The FMT CTR led to a progressive reduction of visceral hypersensitivity in DNBS treated animals which became signi cantly lower after the third transplant cycle (day 28) and consolidated after the fourth one (day 35). The bene cial effect of FMT on post-in ammatory visceral pain caused by DNBS lasted up to 17 days after the treatment interruption (day 49). Starting from day 56, the behavioural response of animals to CRD increase again, returning to the same value as the animals treated with DNBS + vehicle (Fig. 7).

Discussion
The present work highlights the relevance of microbiota in pain signalling from the gut. The FMT from animals affected by visceral pain to healthy rats was su cient to induce visceral hypersensitivity. On the other hand, FMT from healthy donors induced a reduction of persistent visceral pain. A strong association between animals' pain threshold and microbiota composition changes was demonstrated. Indeed, intestinal in ammation as well as gut permeability and gut monoamines were found not to be involved in pain transfer mediated by FMT.
Although the gut microbiota appeared to be clearly involved in visceral pain regulation [3][4][5], the evidence collected so far are circumstantial [16][17][18]. Moreover, little is known on the evolution of microbiota over time after transplantation from animals affected by a disease, like pain, to naïve recipients. Here, we demonstrated a distinct temporal correlation between the animals' sensitivity and changes in the microbiota composition. As suggested by the analysis of the beta-diversity in samples from FMT DNBS animals, major modi cations in the composition and structure of the gut microbiota occurred after the FMT set II (Day 32), correlating with a signi cantly increased response of these animals to CRD. At a later stage (Day 46), when the difference in visceral sensitivity perception disappeared, a less pronounced variation in beta-diversity was observed.
Evaluation of taxonomic pro les con rmed that marked differences between the animals that underwent the FMT CTR or FMT DNBS were identi able on Day 32 at phylum and family level for major members of gut microbiota, including protective microbial taxa known to be associated with gastrointestinal health (i.e. Akkermansiaceae) [19,20]. Akkermansia muciniphila is known to mediate a variety of host functions ranging from metabolism to immune regulation [21][22][23]. Regarding its role in pain syndromes or functional gastrointestinal disorders, A. muciniphila was found to be signi cantly reduced in children with IBS [24] as well as in animal models of post-in ammatory IBS [25]. Recently, Guida et al. reported that pain related to dysbiosis in vitamin D de cient mice were ameliorated by an anandamide congener, in concomitance with increased levels of Akkermansia, Eubacterium and Enterobacteriaceae [26]. However, there is little evidence for a pain-alleviating effect mediated by A. muciniphila. One hypothesis is that SCFAs, like propionate and acetate, both end-products of mucus degradation by Akkermansia, may modulate visceral nociception, as stimulation of the SCFA receptor GPR43 has been shown to decrease visceral pain sensitivity in healthy humans [27,28]. The SCFAs have been associated to human gastrointestinal disorders [29] and proposed as regulators of visceral sensitivity since the activation of their receptors, FFAR2/3 regulates leucocyte functions, such as the production of cytokines (e.g. TNF-a, IL-2, IL-6, and IL-10), eicosanoids and chemokines (e.g. CCL2) [30]. Nevertheless, the role of SCFAs in visceral and somatic pain perception is still the subject of debate: depending on the context the effect of SCFAs signalling can be either analgesic or nociceptive [31]. Clinical evidence has shown that butyrate, a SCFAs found dramatically reduced in the faecal samples of IBS and IBD patients, is highly effective in relieving abdominal pain [32,33]. The signi cant reduction of butyrate in the FMT DNBS group on Day 32 is in line with this evidence. Nevertheless, despite the reduction of butyrate on Day 32, the total amount of SCFAs increased in these animals both on Day 18 and 32 mainly contributed by an overproduction of acetate. Speci c bacteria taxa were found to be negatively correlated with the increased acetate levels uniquely on Day 32, suggesting the presence of a de ned microbiota pro le at this stage. Of note, the recently identi ed bacterial genus Acetatifactor belonging to Lachnospiraceae, one of the most prominent bacterial family being expanded in samples from the FMT DNBS group, and showing strong acetate-producing capabilities [34], resulted overrepresented in FMT DNBS samples ( Figure S4B). However, there are no consistent data in literature reporting a negative impact of acetate on gut physiology. By monitoring the time course modi cations and evolution of the gut microbiota in this model, emerged that the effects of FMT were long-lasting but temporary, demonstrating the presence of a continuous bidirectional in uence between the microbiota and the gut, as previously postulated [35,36]. FMT DNBSinduced visceral hypersensitivity was evident already 24 h after the rst set of FMT, suggesting the presence of pain mediators in the supernatant obtained from the faeces, as supposed in other studies [17,37]. The presence of pain mediators in the faecal medium might explain the acute effect of FMT. By contrast, the long-lasting hyperalgesia observed in these animals is not attributable to the acute stimulation, nor to local intestinal irritation, since colon histological analysis revealed no damage on Day 32 (7 days after the last FMT). These ndings support the idea of an active role of microbiota in pain induction mediated by the FMT. Besides, the apparent longer persistence of visceral hypersensitivity in the AWR test in respect to VMR test, suggested a possible central sensitization to the painful stimulus induced by CRD. Similarly, in the animals treated with DNBS, VMR response was signi cantly decreased after 4 weeks, while the augmented AWR response persisted up to 13 weeks after DNBS injection [38].
A slight increase in gut permeability occurred after the antibiotic treatment, with an increase in the expression of Zo-1, which has been positively correlated with a leaky gut [39], con rming the positive effect of microbiota on gut barrier integrity maintenance [40].
The bacterial community participates in maintaining intestinal homeostasis through the "training" of the immune system and inhibiting growth of pathogens [41]. The analysis of gene expression in the gut con rmed a derangement of cytokines as a result of both the antibiotic treatment and the FMT, but no signi cant correlations were found between the cytokines pro les among the groups and the observed differences in visceral pain.
Then we analysed the effect of FMT on tryptophan metabolism (Table S1-2), considering its relevance in the microbiota-gut-brain axis as well as in the pathophysiology of both IBS and IBDs [42]. A decrease in plasma tryptophan was found in DNBS donors, likely consequent to an increased biosynthesis of serotonin in the gut, as con rmed by the HPLC (Table S3-4). This phenomenon, which is a characteristic consequence of intestinal damages, can directly in uence the neuronal ring of visceral afferents [43,44]. A plasma tryptophan decrease was also detected in the animals underwent FMT and correlated to a massive and long-lasting increase of serotonin in the faeces (Table S6), but not in the gut tissue (Table  S3). An increase of 5-HT in the gut lumen has been recently demonstrated to selectively modulate the colonization of bacteria species in the gut [45]. Nevertheless, no difference was found between the animals receiving the FMT from CTR or from DNBS donors, demonstrating again that 5-HT is not involved in the transfer of pain mediated by FMT DNBS .
The decrease of dopamine consequent to the antibiotic treatment and the FMT (Table S5 and S7) resulted in accordance to previous data reporting an antibiotic-mediated reduction of this neurotransmitter in the gut, where it plays a role in gastrointestinal motility regulation [46].
The anti-hyperalgesic effect obtained by transplanting a healthy microbiota in DNBS animals contributes further to sustain the role of microbiota in pain signalling. Currently, many discrepancies do exist on the therapeutic potential of FMT [47]. So far it has been considered a good protocol to maintain a low number of FMT and supplied by a single donor [48,49]. However, recent clinical studies highlighted the e cacy of a multiple donor and repeated treatment-based therapy in patients affected by UC, chronic intestinal pseudo-obstruction and IBS [50][51][52]. In certain pathological conditions, the interactions between genetic, immunological, and environmental factors might determine a state more resistant to therapeutic microbial manipulation [53]. In the preclinical model of colitis-induced visceral hypersensitivity, we obtained a substantial reduction in pain after 4 cycles of FMT, utilizing different donors. It is likely that the "in ammatory" environment wherein the microbiota is transplanted may pose as an obstacle to the engraftment of some species of microbiota compared to others. In this case, the repetition of the FMT could "force" the host to accept the newly introduced microbiota. These ndings suggest the possibility to enhance the therapeutic e cacy of FMT by developing different protocols according to the speci c disorder to be treated.

Conclusions
The results obtained in vivo strengthen the hypothesis of an active contribution of gut microbiota to visceral pain induction and persistence and encourage the continued investigation into the mechanisms by which the microbiota modulates visceral sensitivity, in order to make it a suitable target for the treatment of abdominal pain.

Animals
For all the experiments described below, Male Sprague-Dawley rats (Envigo, Varese, Italy) weighing approximately 220-250 g at the beginning of the experimental procedure, were used. Animals were housed in CeSAL (Centro Stabulazione Animali da Laboratorio, University of Florence) and used at least 1 week after their arrival. Four rats were housed per cage (size 26 × 41 cm); animals were fed a standard laboratory diet and tap water ad libitum, and kept at 23 ± 1 °C with a 12 h light/dark cycle, light at 7 a.m. . Formal approval to conduct the described experiments was obtained from the Animal Subjects Review Board of the University of Florence. Experiments involving animals have been reported according to ARRIVE guidelines [54]. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Induction of colitis
Colitis was induced in accordance with the method described previously by Antonioli et al. [55] with minor changes. In brief, during a short anaesthesia with iso urane (2%), 30 mg of 2,4-dinitrobenzenesulfonic acid (DNBS) in 0.25 ml of 50% ethanol was administered intrarectally via a polyethylene PE-60 catheter inserted 8 cm proximal to the anus. Control rats received 0.25 ml of saline solution.

Faecal Microbiota Transplantation (FMT) study design
In the rst experimental set, rats were randomized into the following treatment groups (n = 10): control (vehicle, no antibiotic treatment, no FMT); abx + vehicle (antibiotic treatment followed by vehicle administration); abx + FMT CTR (antibiotic treatment followed by FMT from controls); abx + FMT DNBS (antibiotic treatment followed by FMT from DNBS treated animals). The animals were singly housed and underwent the following antibiotic/antifungal regimen to prepare them to the FMT: day 0-6 rats received a daily oral gavage of amphotericin B (1 mg kg − 1 ) and metronidazole (100 mg kg − 1 ) while an antibiotic mix (ceftazidime 1 g L − 1 , vancomycin 0.5 g L − 1 and neomicin 1 g L − 1 ) was added to the autoclaved drinking water and changed every two days. On day 7, 24 h after the interruption of the antibiotic treatment, the animals underwent the FMT procedure. FMT was daily performed on days 7-11 (Set I) and on days 20-24 (Set II). Behavioural tests were performed at the end of the antibiotic treatment (before starting the FMT), 24 h and 7 days after each FMT set and once week after the last treatment. Effects of the antibiotic/antifungal treatments on the gut microbiota were evaluated by a culture-based analysis for For the FMT, faecal material was collected from controls (n = 3) and DNBS treated animals (n = 3) (between 14 and 21 days after the intra-rectal injection of the in ammatory agent) and placed into tubes containing sterile saline solution, tubes were left on ice for 60 min and later homogenized for 2 min on ice using a hand-held pellet pestle device with sterile, re-usable pestles. When fully homogenized, the suspended pellets were directly utilized for the FMT procedure. FMT was carried out via oral gavage with a faecal suspension (50 mg ml − 1 ) in a nal volume of 3 ml.
In the second experimental set, rats were randomized into the following treatment groups (n = 5): control (vehicle); DNBS + vehicle (intrarectal injection of DNBS 30 mg followed by vehicle administration); DNBS + FMT CTR (intrarectal injection of DNBS 30 mg followed by FMT from controls). Colitis was induced in the animals by the intra rectal injection of DNBS (30 mg in 0.25 mL EtOH 50%) on day 1. A control group was intra-rectally administered with saline solution. Seven day after DNBS injection the animals were split into 2 groups, respectively receiving the vehicle or the naïve controls-derived faecal microbiota suspensions (FMT CTR ). FMT was performed for 5 consecutive days/week and the same protocol was repeated for 4 weeks (I-IV set of FMT): on days 7-11 (Set I), 14-18 (Set II), 21-25 (Set III) and 28-32 (Set IV) after DNBS injection. Behavioural tests were carried out 7 days after DNBS injection (before starting FMT), 3 days after each FMT set and once week after the last treatment. For the FMT, faecal material was collected from control naïve animals (n = 3) and placed into tubes containing sterile saline solution, tubes were left on ice for 60 min and later homogenized for two min on ice using a hand-held pellet pestle device with sterile, re-usable pestles. When fully homogenized, the suspended pellets were directly utilized for the FMT procedure. FMT was carried out via oral gavage with a faecal suspension (50 mg ml − 1 ) in a nal volume of 3 ml.

Assessment of visceral sensitivity by Viscero Moto Response (VMR)
The visceromotor response (VMR) to colorectal balloon distension were used as objective measure of visceral sensitivity in animals. Two EMG electrodes were sutured into the external oblique abdominal muscle under deep anaesthesia and exteriorised dorsally [56]. VMR assessment were carried out under light anaesthesia (iso urane 2%) as previously reported [57]. A lubricated latex balloon (length: 4.5 cm), assembled to an embolectomy catheter and connected to a syringe lled with water was used to perform colo-rectal distension. The balloon was inserted into the colon and positioned 6.5 cm from the anus and was lled with increasing volumes of water (0.5, 1, 2, 3 mL). The electrodes were relayed to a data acquisition system and the corresponding EMG signal consequent to colo-rectal stimulation were

Assessment of visceral sensitivity by Abdominal Withdrawal Re ex (AWR)
The behavioural responses to CRD were assessed in the animals by measuring the Abdominal Withdrawal Re ex (AWR), a semi-quantitative score described previously in conscious animals [58].
Brie y, rats were anesthetized with iso urane, and a lubricated latex balloon (length: 4.5 cm), attached to polyethylene tubing, assembled to an embolectomy catheter and connected to a syringe lled with water was inserted through the anus into the rectum and descending colon of adult rats. The tubing was taped to the tail to hold the balloon in place. Then rats were allowed to recover from the anaesthesia for 30 min.
AWR measurement consisted of visual observation of animal responses to graded CRD (0.5, 1, 2, 3 mL) blinded observers who assigned AWR scores: No behavioral response to colorectal distention (0); Immobile during colorectal distention and occasional head clinching at stimulus onset (1); Mild contraction of the abdominal muscles but absence of abdomen lifting from the platform (2); Observed strong contraction of the abdominal muscles and lifting of the abdomen off the platform (3); Arching of the body and lifting of the pelvic structures and scrotum (4). The time elapsed between two consecutive distension was 5 min.

Pro ling of gut microbiota
Fecal pellets (50 mg) and fecal suspensions (volume equivalent to 50 mg) were processed for the total DNA extraction using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany). Next generation sequencing of 16S ribosomal RNA amplicons (V3-V4 regions) was performed using the Illumina MiSeq platform, according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Part # 15044223 Rev. B; URL: http://www.illumina.com/content/dam/illuminasupport/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf), following a 2 × 300 bp paired-end approach. Sequencing results were analyzed using the QIIME 2 suite (Quantitative Insights Into Microbial Ecology) [59]. Brie y, following raw reads denoising (i.e. error correction, removal of chimeric and singleton sequences, join of denoised paired-end reads) using DADA2 [60] for each sample amplicon sequence variants (ASVs) were inferred. Taxonomic classi cation of dereplicated ASV was performed using a Naive Bayes classi er trained on the SILVA 16S reference database (release 132) (https://www.arb-silva.de/documentation/release-132/). Microbial diversity was estimated by evaluating alpha-diversity (Shannon index) and beta-diversity (Bray-Curtis) metrics using speci c tools implemented in the QIIME 2 pipeline. The 16S rRNA sequence data has been  Declarations Figure 1 Effect of antibiotic treatment and FMT from DNBS-treated animals on visceral sensitivity of naïve recipients. As shown in the scheme, rats were treated, with a combination of antibiotics for 7 days; control group was treated with vehicle. On day 7 the antibiotics (abx)-treated animals were divided into 3 groups, FMT from CTR donors, FMT from DNBS donors or vehicle were respectively administered per os for 5 consecutive days. One week after the administrations were repeated. Behavioural tests were performed at      Therapeutic effect of FMT on DNBS-induced post-in ammatory visceral pain. As shown in the scheme, rats were intra-rectally injected with DNBS (30 mg in 0.25 mL EtOH 50%); on day 7 DNBS-animals were divided into 2 groups respectively administered with the vehicle or the FMT from CTR donors per os for 5 consecutive days. The FMT set was weekly repeated for 4 times. Behavioral tests were performed at the end of the antibiotic treatment, 3 days after each cycle of FMT and once week afterwards. Visceral