Schmidt, Bradley A.

The human gastrointestinal (GI) tract is home to trillions of microorganisms. There is dynamic interaction between the gut microbiota and the human host, but how gut microbiota modulates GI physiology is largely unknown. A recent study from our laboratory showed that human gut microbiota increases host serotonin (5‐hydroxytryptamine, 5‐HT) biosynthesis. 5‐HT is an important neurotransmitter and a paracrine messenger in the GI tract that increases intestinal secretion primarily through 5‐HT 3 and 5‐HT 4 receptors. In this study we investigated whether gut microbiota modulate 5‐HT 3 R and 5‐HT 4 R expression and host secretory response to 5‐HT. Methods We used proximal colon mucosa‐submucosa preparations from germ free (GF) and humanized (ex‐GF colonized with human bacteria for 4–5 weeks; HM) male mice. The change in short circuit current (ΔIsc) was determined in response to increasing concentrations of 5‐HT (0.003–300μM) on the submucosal side either alone or in the presence of 5‐HT 3 R antagonist ondansetron (100nM) in an Ussing chamber. Receptor expression was quantified using rt‐PCR (normalized to L32) and western blot (normalized to GAPDH). Immunohistochemistry (IHC) was used to determine localization of 5‐HT 3 R in the paraformaldehyde‐fixed 10μm sections from proximal colonic epithelium. Differences between groups were tested using Mann‐Whitney U (Mdn; IQ range) with P ‐value lower than 0.05 considered significant. Results 5‐HT induced ΔIsc was significantly higher in GF than HM mice (218; 184–274 vs 163; 132–182 μA/cm 2 , n=6, P = 0.04). To determine if this change was due to an alteration in 5‐HTR, we quantified receptor expression. We found significantly higher 5‐HT 3 R mRNA expression (1.82; 1.08–2.80 vs 1.00; 0.33–1.38, n=8–9, P = 0.046) in GF compared to HM mice, but no change in 5‐HT 4 R expression. 5‐HT 3 R protein expression was also significantly higher in GF mice compared to HM (4.62; 1.06–6.14 vs 1.00; 1–1.3, n= 11–12, P = 0.01) mice. There was a significantly higher ΔIsc in GF compared to HM (87.1; 51.7–149 vs 37.0; 19.5–44 μA/cm 2 , n=5, P = 0.02) mice, following application of 2‐methyl 5‐hydroxytryptamine hydrochloride (selective 5‐HT 3 R agonist) suggesting increased receptor expression augments secretory response. Furthermore, ondansetron significantly blocked 5‐HT induced maximum ΔIsc in GF mice (201; 189–260 vs 117.6; 92–149 μA/cm 2 , n=6–8, P = 0.001) but not in HM mice (163; 132–182 vs 120; 107–150 μA/cm 2 , n=6, P = 0.24). To determine the expression of 5‐HT 3 R in mouse colon mucosa‐submucosa we used 5‐HT 3 –tagged GFP mice. IHC identified 5‐HT 3 R‐GFP positive cells in the mouse colonic epithelium. To ascertain if the significant difference in 5‐HT‐induced ΔIsc between GF and HM mice (188; 152–200 vs 125; 99–139 μA/cm 2 , n=5, P = 0.008) is driven by epithelial 5‐HT 3 R, we measured the response in the presence of TTX (block neuronal transmission). TTX (500 nM) had no effect of ondansetron inhibition of 5‐HT‐induced ΔIsc, suggesting a role for epithelial 5HT 3 R. Conclusion Gut microbial colonization decreases host secretory response to 5‐HT by downregulating 5‐HT 3 R expression. Epithelial 5‐HT 3 R may be important in gut microbiota modulation of intestinal secretion Support or Funding Information NIH K08 DK

Increased amino acids in the dysbiotic gut influences susceptibility to Clostridioides difficile infection in mice and humans.

Serotonin [5-hydroxytryptamine (5-HT)], an important neurotransmitter and a paracrine messenger in the gastrointestinal tract, regulates intestinal secretion by its action primarily on 5-HT3and 5-HT4receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study, we determine whether human-derived gut microbiota affects host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age-matched Swiss Webster germ-free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short-circuit current (Δ Isc) in GF compared with HM mice. Additionally, 5-HT3receptor mRNA and protein expression was significantly higher in GF compared with HM mice. Ondansetron, a 5-HT3receptor antagonist, inhibited 5-HT-evoked Δ Iscin GF mice but not in HM mice. Furthermore, a 5-HT3receptor-selective agonist, 2-methyl-5-hydroxytryptamine hydrochloride, evoked a significantly higher Δ Iscin GF compared with HM mice. Immunohistochemistry in 5-HT3A-green fluorescent protein mice localized 5-HT3receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT-evoked Δ Iscbetween GF and HM mice persisted in the presence of tetrodotoxin (TTX) but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT3receptor mRNA in GF mouse colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT3receptor expression via acetate production. Epithelial 5-HT3receptor may function as a mediator of gut microbiota-driven change in intestinal secretion.NEW & NOTEWORTHY We found that gut microbiota alters serotonin (5-HT)-evoked intestinal secretion in a 5-HT3receptor-dependent mechanism and gut microbiota metabolite acetate alters 5-HT3receptor expression in colonoids.View this article's corresponding video summary at https://www.youtube.com/watch?v=aOMYJMuLTcw&feature=youtu.be .

SummaryBackgroundClostridium difficile infection (CDI) may not respond to initial therapy and frequently recurs, but predictors of response and recurrence are inconsistent. The impact of specific alterations in the gut microbiota determining treatment response and recurrence in patients with CDI is unknown.AimTo assess microbial signatures as predictors of treatment response and recurrence in CDI.MethodsPre‐treatment stool samples and clinical metadata including outcomes were collected prospectively from patients with their first CDI episode. Next generation 16s rRNA sequencing using MiSeq Illumina platform was performed and changes in microbial community structure were correlated with CDI outcomes.ResultsEighty‐eight patients (median age 52.7 years, 60.2% female) were included. Treatment failure occurred in 12.5% and recurrence after response in 28.5%. Patients who responded to treatment had an increase in Ruminococcaceae, Rikenellaceae, Clostridiaceae, Bacteroides, Faecalibacterium and Rothia compared to nonresponders. A risk‐index built from this panel of microbes differentiated responders (mean 0.07 ± 0.24) from nonresponders (0.52 ± 0.42; P = 0.0002). Receiver operating characteristic (ROC) curve demonstrated that risk‐index was a strong predictor of treatment response with an area under the curve (AUC) of 0.85. Among clinical parameters tested, only proton pump inhibitor use predicted recurrent CDI (OR 3.75, 95% CI 1.27–11.1, P = 0.01). Patients with recurrent CDI had statistically significant increases in Veillonella, Enterobacteriaceae, Streptococci, Parabacteroides and Lachnospiraceae compared to patients without recurrence and a risk index was able to predict recurrence (AUC = 0.78).ConclusionGut microbiota signatures predict treatment response and recurrence potentially, allowing identification of patients with Clostridium difficile infection that may benefit from early institution of alternate therapies.