A 3D intestinal tissue model supports Clostridioides difficile germination, colonization, toxin production and epithelial damage

Abstract

Endospore-forming Clostridioides difficile is a causative agent of antibiotic-induced diarrhea, a major nosocomial infection. Studies of its interactions with mammalian tissues have been hampered by the fact that C. difficile requires anaerobic conditions to survive after spore germination. We recently developed a bioengineered 3D human intestinal tissue model and found that low O conditions are produced in the lumen of these tissues. Here, we compared the ability of C. difficile spores to germinate, produce toxin and cause tissue damage in our bioengineered 3D tissue model versus in a 2D transwell model in which human cells form a polarized monolayer. 3D tissue models or 2D polarized monolayers on transwell filters were challenged with the non-toxin producing C. difficile CCUG 37787 serotype X (ATCC 43603) and the toxin producing UK1 C. difficile spores in the presence of the germinant, taurocholate. Spores germinated in both the 3D tissue model as well as the 2D transwell system, however toxin activity was significantly higher in the 3D tissue models compared to the 2D transwells. Moreover, the epithelium damage in the 3D tissue model was significantly more severe than in 2D transwells and damage correlated significantly with the level of toxin activity detected but not with the amount of germinated spores. Combined, these results show that the bioengineered 3D tissue model provides a powerful system with which to study early events leading to toxin production and tissue damage of C. difficile with mammalian cells under anaerobic conditions. Furthermore, these systems may be useful for examining the effects of microbiota, novel drugs and other potential therapeutics directed towards C. difficile infections.

Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

“…While both their 3D model and our models have epithelial and fibroblast cells, albeit with different scaffolds, a key difference is the ability to control the apical and basolateral side environments using the VDC, which may be important when performing sensitive molecular studies. Similar to Shaban et al (2018), we show bacterial replication up to 48 h, although we also track bacteria that adhere to the epithelial layer. Additionally, we have a nanofiber scaffold incorporated between the epithelial and fibroflast layers, which creates a porous architecture similar to the basement membrane underlying the gut epithelium, along with myofibroblast cells in the basal layer.…”

“…Three-dimensional models are known to promote more in vivo -like cell proliferation, growth differentiation, and cell-to cell contact (Kook et al, 2017). A recent study reported a 3D intestinal tissue model for C. difficile , where the authors demonstrated that C. difficile toxin activity was higher in comparison to a 2D transwell model (Shaban et al, 2018). They report that spores can germinate into vegetative cells within this model and that vegetative cells can survive up to 48 h, although the ability of C. difficile spores to germinate was similar in both their models.…”

Advances in modelling the human microbiome–gut–brain axis in vitro

“…Reproduced from [ 55 ], under the Creative Commons license; ( C ) Schematic of C. difficile infection in the 3D scaffold tissue model (left) and scanning electron microscopy of uninfected (left column) and infected with UK1 C. difficile 3D scaffolds (right column) at 4, 24 and 48 h (right). Adapted from [ 23 ].…”

A review of co-culture models to study the oral microenvironment and disease

“…Links between the gut microbiome and obesity, diabetes, liver disease, cancer, and neurodegenerative diseases [ 75 , 76 ] have now been established, which has driven considerable growth in this research area. Similar approaches to the co-culture models described herein have been utilised in this field, including monolayer/planktonic cultures [ 77 – 80 ] and 3D/planktonic cultures [ 81 , 82 ]. Reported methods to generate an in vitro environment that better represents the in vivo surroundings of the digestive tract include bioreactors [ 79 ], 3D organoid cultures [ 83 , 84 ], and organ-on-a-chip systems [ 85 ], all of which are described in detail by a review published in 2017 [ 78 ].…”