Spatial and temporal modulation of enterotoxigenic E. coli H10407 pathogenesis and interplay with microbiota in human gut models

Abstract

Background Enterotoxigenic Escherichia coli (ETEC) substantially contributes to the burden of diarrheal illnesses in developing countries. With the use of complementary in vitro models of the human digestive environment, TNO gastrointestinal model (TIM-1), and Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME), we provided the first detailed report on the spatial-temporal modulation of ETEC H10407 survival, virulence, and its interplay with gut microbiota. These systems integrate the main physicochemical parameters of the human upper digestion (TIM-1) and simulate the ileum vs ascending colon microbial communities and luminal vs mucosal microenvironments, captured from six fecal donors (M-SHIME). Results A loss of ETEC viability was noticed upon gastric digestion, while a growth renewal was found at the end of jejunal and ileal digestion. The remarkable ETEC mucosal attachment helped to maintain luminal concentrations above 6 log10 mL−1 in the ileum and ascending colon up to 5 days post-infection. Seven ETEC virulence genes were monitored. Most of them were switched on in the stomach and switched off in the TIM-1 ileal effluents and in a late post-infectious stage in the M-SHIME ascending colon. No heat-labile enterotoxin production was measured in the stomach in contrast to the ileum and ascending colon. Using 16S rRNA gene-based amplicon sequencing, ETEC infection modulated the microbial community structure of the ileum mucus and ascending colon lumen. Conclusions This study provides a better understanding of the interplay between ETEC and gastrointestinal cues and may serve to complete knowledge on ETEC pathogenesis and inspire novel prophylactic strategies for diarrheal diseases.

Multi-targeted properties of the probiotic saccharomyces cerevisiae CNCM I-3856 against enterotoxigenic escherichia coli (ETEC) H10407 pathogenesis across human gut models

“… 64 The TIM-1 system was programmed based on in vivo data to simulate the physicochemical digestive conditions from the stomach to the ileum, encountered in a healthy adult. 65 The system does not include microbial ecosystem, contrary to the M-SHIME (section below). Three conditions were tested in TIM-1 ( Figure 1a ): (i) control experiment consisted of feeding the stomach compartment with mineral water (200 mL) experimentally contaminated with ETEC (7.5 log 10 CFU mL −1 ); (ii) probiotic control condition consisted in the administration of the yeast S. cerevisiae alone (7.5 log 10 CFU mL −1 ); and (iii) probiotic treatment condition consisted in the co-administration of ETEC (7.5 log 10 CFU mL −1 ) and the probiotic yeast (7.5 log 10 CFU mL −1 ).…”

“…67 All vessels were flushed with N 2 immediately after inoculation to generate anaerobic conditions. In addition, the mucosal environment was mimicked in both ileum and ascending colon compartments, through the incorporation of microcosms (AnoxKaldnes K1 carrier, Lund, Sweden) coated with type III porcine mucin-agar (Sigma͐–aldrich, St. Louis, US), instead of type II mucin, as described by Van den Abbeele et al 68 Functioning of the system, mucin carrier replacement and media composition have been presented in Roussel et al 65 …”

“…Prior to the challenge at day 13, ETEC and S. cerevisiae were pre-digested 3 h under batch conditions, to reproduce the gastro-jejunal digestion of a glass of mineral water, where physicochemical conditions were close to those found in TIM-1 (without nutritional medium, under aerobic conditions). 65 SHIME suspensions from ileum and ascending colon vessels were sampled every two days for SCFA analysis and ethanol production. DNA, RNA samples, and supernatants for ELISA measurement were collected at different hours from day 13, and until 29 h post-infection and stored as previously explained for TIM-1 samples.…”

“…One aliquot was stained with 50 μM propidium monoazide (PMA, Interchim, Montluçon, France). 65 The qPCR procedure was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems) with 16S Enterobacteriaceae primers for TIM-1 and gspD primers for M-SHIME, as previously described. 65 Probiotic yeasts were plated onto Sabouraud agar (BD Difco, Waltham, USA), supplemented with chloramphenicol (50 mg L −1 ) (Sigma, St Louis, USA) and incubated at 30°C for 48 h. All statistical analyses were performed in R studio, version 3.6.1 (R Core Team, 2019), using PMCMR package, version 4.3.…”

“…Total RNA was extracted from TIM-1 and SHIME digestive samples using the TRIzol® method (Invitrogen, Thermo Fisher Scientific, Waltham, USA) according to the author recommendation. 65 DNAse treatment and RNA quality control were performed respectively, according to the manufacturer’s recommendation (TURBO DNA-free TM , Invitrogen, Thermo Fisher Scientific, Waltham, USA) and Roussel et al 65 RT-qPCR was used to analyze the expression of seven virulence genes encoding for enterotoxins ( eltB and estP ), enterotoxin release ( leoA and tolC ), and adhesins ( cfa/Ib, tia, fimH ). 65 Non-parametric test was used to compare the log 2 fold change in gene expression under control versus probiotic conditions using Nemenyi post-hoc test conducted following significant results for the Friedman test.…”