Use of the Dynamic TIM-1 Model for an In-Depth Understanding of the Survival and Virulence Gene Expression of Shiga Toxin-Producing Escherichia coli in the Human Stomach and Small Intestine

International audienceDue to obvious ethical and technical reasons, it remains very difficult to evaluate the survival and expression of virulence genes of food-borne pathogens, such as Shiga toxin-producing Escherichia coli (STEC) in the human gastrointestinal tract. Here, we describe the use of the dynamic TNO (Toegepast Natuurwetenschappelijk Onderzoek) gastrointestinal model (TIM-1) as a powerful in vitro tool to obtain the kinetics of STEC survival by plate counting, the regulation of major virulence genes by RT-qPCR, and the production of Shiga toxins by ELISA, in the human stomach and small intestine. The gut model was adapted in order that in vitro digestions were performed both under adult and child digestive conditions, specific at risk populations for STEC infections.STEC Survival Virulence genes Shiga toxins Human GIT In vitro gut models TIM-

Evidence of gene transfer via Integrative and Conjugative Elements (ICEs) between bacteria in the human small intestine environment using the dynamic in vitro system TIM-1.

International audienceConsumption of food brings large amounts of various bacteria able to interact and exchange genes with the host ones. Gene acquisition can confer novel properties to bacteria (antibiotic resistance, catabolic properties…) which may threat the equilibrium of digestive microbiota or lead to the emergence of new pathogens.Conjugation is the main mechanism of gene acquisition. Gene transfer can occur between strains belonging to the same species but also between very distantly related bacteria. Several classes of mobile genetic elements can be transferred by conjugation, in particular chromosomal elements called Integrative and Conjugative Elements (ICEs), a widespread but poorly known class of elements.To give a first insight on the incidence of gene transfers mediated by ICEs in the human digestive ecosystem, we evaluated their occurrence between dietary and intestinal commensal bacteria during digestion, using the TIM-1 model able to mimic gastric and small intestine environment. Different pairs of ICE donors (Streptococcus thermophilus, S. salivarius) and receptors (S. thermophilus, S. salivarius, Enterococcus faecalis) were immobilized on alginate/agar/chitosan beads and maintained in the jejunum and the ileum of the TIM-1 during milk digestion. Generation of transconjugants (receptors integrating ICE) was evaluated after an exposure of 5h to those conditions, and compared to exposure to undigested milk and M17 broth.Among the 11 pairs tested, 4 gave transconjugants during in vitro digestion whereas all generated conjugates in milk and/or M17. Remarkably, two pairs involved transfer between different genera (S. thermophilus x E. faecalis), whereas the other two involved the same species (S. thermophilus x S. thermophilus). Our study highlights that gene acquisition via transfer of ICEs should occur relatively frequently in the human digestive environment. Moreover, the use of dynamic in vitro models such as the TIM-1 gives a good alternative to in vivo study, to explore such phenomenon easily without ethical constraints

Impact of physicochemical parameters of the digestive tract on Cryptosporidium parvum infection

International audienceCryptosporidium parvum is responsible for a zoonotic disease affecting both human health and livestock. The parasite infects its host through the oral route and develops in ileal epithelial cells, leading to acute and sometimes lethal diarrhoea. The severity of cryptosporidiosis is closely related to the immune status of its host, young ruminants, infants, and immunocompromised individuals being more susceptible. Other non-immune-related factors, such as digestive physicochemical properties, can also vary widely with age but their contribution to infection establishment in young individuals has never been investigated. In vitro digestion models have been designed and validated to study the fate of orally ingested substances while closely mimicking the physiological processes occurring during digestion. Among the available systems, the dynamic and multicompartmental TNO gastrointestinal model (TIM-1) is currently the most complete simulator of the human upper gastrointestinal tract. The TIM-1 was used for a comparative study of C. parvum infection under adult and young children (from 6 months to 2 years) digestive conditions. The TIM-1 was programmed to reproduce both physicochemical digestive conditions upon simulated ingestion of a glass of mineral water, which was experimentally contaminated with C. parvum oocysts. The parasite excystation kinetics throughout the course of the in vitro digestion was determined by flow cytometry analysis. Parasite invasion ability was assessed after reinoculation of sporozoites collected from the TIM-1 onto HCT-8 cells. A luciferase reporter gene assay was also used to follow sporozoite activity throughout the digestive process. Our data show that the parasite excystation rate is almost maximal in the duodenal compartment, one hour after the beginning of digestion in the TIM-1. However, a higher number of parasites reaches the distal ileal compartment while protected in their oocyst shell upon simulation of child compared to adult digestive conditions. This suggests that a lower amount of sporozoites is released in the small intestine in children, nevertheless, the luciferase activity expressed by these free stages in the ileal compartment is significantly higher in children compared to adults. Invasion assay performed on HCT-8 cells suggests that only sporozoites collected in the ileum after three hours of digestion are able to invade host cells.Our study is the first one exploring the impact of different digestive conditions on Cryptosporidium using a sophisticated gastrointestinal model. A global transcriptome analysis by RNA-Seq is also being performed on samples collected during in vitro digestion to identify parasite genes that are differentially expressed under child and adult digestive conditions

Impact of physicochemical parameters of child or adult digestive tract on Cryptosporidium parvum infection

National audienceCryptosporidium parvum is responsible for a zoonotic disease affecting both human health and livestock. The parasite infects its host through the oral route and develops in ileal epithelial cells, leading to acute and sometimes lethal diarrhoea. The severity of cryptosporidiosis is closely related to the immune status of its host, young ruminants, infants, and immunocompromised individuals being more susceptible. Other non-immune-related factors, such as digestive physicochemical properties, can also vary widely with age but their contribution to infection establishment in young individuals has never been investigated. In vitro digestion models have been designed and validated to study the fate of orally ingested substances while closely mimicking the physiological processes occurring during digestion. Among the available systems, the dynamic and multicompartmental TNO gastrointestinal model (TIM-1) is currently the most complete simulator of the human upper gastrointestinal tract. The TIM-1 was used for a comparative study of C. parvum infection under adult and young children digestive conditions. The TIM-1 was programmed to reproduce both physicochemical digestive conditions upon simulated ingestion of a glass of mineral water, which was experimentally contaminated with C. parvum oocysts. The parasite excystation kinetics throughout the course of the in vitro digestion was determined by flow cytometry analysis. Parasite invasion ability was assessed after reinoculation of sporozoites collected from the TIM-1 onto HCT-8 cells. A luciferase reporter gene assay was also used to follow sporozoite activity throughout the digestive process. Our data show that the parasite excystation rate is almost maximal in the duodenal compartment, one hour after the beginning of digestion in the TIM-1. However, a higher number of parasites reaches the distal ileal compartment while protected in their oocyst shell upon simulation of child compared to adult digestive conditions. This suggests that a lower amount of sporozoites is released in the small intestine in children, nevertheless, the luciferase activity expressed by these free stages in the ileal compartment is significantly higher in children compared to adults. Invasion assay performed on HCT-8 cells suggests that only sporozoites collected in the ileum after three hours of digestion are able to invade host cells.Our study is the first one exploring the impact of different digestive conditions on Cryptosporidium using a sophisticated gastrointestinal model. A global transcriptome analysis by RNA-Seq is also being performed on samples collected during in vitro digestion to identify parasite genes that are differentially expressed under child and adult digestive conditions

Impact of physicochemical parameters of the digestive tract on Cryptosporidium parvum infection

International audienceCryptosporidium parvum is responsible for a zoonotic disease affecting both human health and livestock. The parasite infects its host through the oral route and develops in ileal epithelial cells, leading to acute and sometimes lethal diarrhoea. The severity of cryptosporidiosis is closely related to the immune status of its host, young ruminants, infants, and immunocompromised individuals being more susceptible. Other non-immune-related factors, such as digestive physicochemical properties, can also vary widely with age but their contribution to infection establishment in young individuals has never been investigated. In vitro digestion models have been designed and validated to study the fate of orally ingested substances while closely mimicking the physiological processes occurring during digestion. Among the available systems, the dynamic and multicompartmental TNO gastrointestinal model (TIM-1) is currently the most complete simulator of the human upper gastrointestinal tract. The TIM-1 was used for a comparative study of C. parvum infection under adult and young children (from 6 months to 2 years) digestive conditions. The TIM-1 was programmed to reproduce both physicochemical digestive conditions upon simulated ingestion of a glass of mineral water, which was experimentally contaminated with C. parvum oocysts. The parasite excystation kinetics throughout the course of the in vitro digestion was determined by flow cytometry analysis. Parasite invasion ability was assessed after reinoculation of sporozoites collected from the TIM-1 onto HCT-8 cells. A luciferase reporter gene assay was also used to follow sporozoite activity throughout the digestive process. Our data show that the parasite excystation rate is almost maximal in the duodenal compartment, one hour after the beginning of digestion in the TIM-1. However, a higher number of parasites reaches the distal ileal compartment while protected in their oocyst shell upon simulation of child compared to adult digestive conditions. This suggests that a lower amount of sporozoites is released in the small intestine in children, nevertheless, the luciferase activity expressed by these free stages in the ileal compartment is significantly higher in children compared to adults. Invasion assay performed on HCT-8 cells suggests that only sporozoites collected in the ileum after three hours of digestion are able to invade host cells.Our study is the first one exploring the impact of different digestive conditions on Cryptosporidium using a sophisticated gastrointestinal model. A global transcriptome analysis by RNA-Seq is also being performed on samples collected during in vitro digestion to identify parasite genes that are differentially expressed under child and adult digestive conditions

Impact of physicochemical parameters of child or adult digestive tract on Cryptosporidium parvum infection

National audienceCryptosporidium parvum is responsible for a zoonotic disease affecting both human health and livestock. The parasite infects its host through the oral route and develops in ileal epithelial cells, leading to acute and sometimes lethal diarrhoea. The severity of cryptosporidiosis is closely related to the immune status of its host, young ruminants, infants, and immunocompromised individuals being more susceptible. Other non-immune-related factors, such as digestive physicochemical properties, can also vary widely with age but their contribution to infection establishment in young individuals has never been investigated. In vitro digestion models have been designed and validated to study the fate of orally ingested substances while closely mimicking the physiological processes occurring during digestion. Among the available systems, the dynamic and multicompartmental TNO gastrointestinal model (TIM-1) is currently the most complete simulator of the human upper gastrointestinal tract. The TIM-1 was used for a comparative study of C. parvum infection under adult and young children digestive conditions. The TIM-1 was programmed to reproduce both physicochemical digestive conditions upon simulated ingestion of a glass of mineral water, which was experimentally contaminated with C. parvum oocysts. The parasite excystation kinetics throughout the course of the in vitro digestion was determined by flow cytometry analysis. Parasite invasion ability was assessed after reinoculation of sporozoites collected from the TIM-1 onto HCT-8 cells. A luciferase reporter gene assay was also used to follow sporozoite activity throughout the digestive process. Our data show that the parasite excystation rate is almost maximal in the duodenal compartment, one hour after the beginning of digestion in the TIM-1. However, a higher number of parasites reaches the distal ileal compartment while protected in their oocyst shell upon simulation of child compared to adult digestive conditions. This suggests that a lower amount of sporozoites is released in the small intestine in children, nevertheless, the luciferase activity expressed by these free stages in the ileal compartment is significantly higher in children compared to adults. Invasion assay performed on HCT-8 cells suggests that only sporozoites collected in the ileum after three hours of digestion are able to invade host cells.Our study is the first one exploring the impact of different digestive conditions on Cryptosporidium using a sophisticated gastrointestinal model. A global transcriptome analysis by RNA-Seq is also being performed on samples collected during in vitro digestion to identify parasite genes that are differentially expressed under child and adult digestive conditions

Exposure to polyethylene microplastics alters immature gut microbiome in an infant in vitro gut model

International audienceInfants are characterized by an immaturity of the gut ecosystem and a high exposure to microplastics (MPs) through diet, dust and suckling. However, the bidirectional interactions between MPs and the immature infant intestinal microbiota remain unknown. Our study aims to investigate the impact of chronic exposure to polyethylene (PE) MPs on the gut microbiota and intestinal barrier of infants, using the new Toddler mucosal Artificial Colon coupled with a co-culture of epithelial and mucus-secreting cells. Gut microbiota composition was determined by 16S metabarcoding and microbial activities were evaluated by gas, short chain fatty acid and volatolomics analyses. Gut barrier integrity was assessed via evaluation of intestinal permeability, inflammation and mucus synthesis. Exposure to PE MPs induced gut microbial shifts increasing α-diversity and abundance of potentially harmful pathobionts, such as Dethiosulfovibrionaceae and Enterobacteriaceae. Those changes were associated to butyrate production decrease and major changes in volatile organic compounds profiles. In contrast, no significant impact of PE MPs on the gut barrier, as mediated by microbial metabolites, was reported. For the first time, this study indicates that ingestion of PE MPs can induce perturbations in the gut microbiome of infants. Next step would be to further investigate the potential vector effect of MPs