Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

Abstract

Interactions of anaerobic gut bacteria, such as Clostridium difficile , with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei , and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei . Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host–anaerobe and pathogen–commensal interaction studies in vitro .

Clostridioides difficile exploits toxin-mediated inflammation to alter the host nutritional landscape and exclude competitors from the gut microbiota

“…suggesting that the inflammation induced by wild type C. difficile in our mouse model selects against a Bacteroidaceae population expansion. In support of this, negative associations between C. difficile and members of the Bacteroidaceae have been reported in human studies, as well as in vitro, and in mouse models of CDI[59][60][61][62][63] . Further work is necessary to identify which mediator(s) of host inflammation are responsible for the restriction of the Bacteroidaceae in our model of CDI.…”

Dissecting individual pathogen-commensal interactions within a complex gut microbiota community

“…B. dorei and C. difficile were cultured overnight in brain heart infusion (BHI) media (Sigma Aldrich) supplemented with 0.5 mg/ml yeast extract (Fisher Scientific) and 0.001 mg/ml L-cysteine (BHIS) 38 . After which, cultures were diluted down to 0.1 OD and added to a 24-well polystyrene tissue culture treated plate.…”

“…Dilutions were plated on BHIS with C. difficile supplement (Oxoid). C. difficile and B. dorei have two distinct colony morphologies 38 , which allowed us to differentiate between Bacteroides and C. difficile and quantitate each species…”

A novel 3D in vitro model of the human gut microbiota

“…Burkholderia terricola adhesion on a poly(ε-caprolactone) nanofiber mats was widely studied by De Cesare and colleagues 17 . Also, Anonye and coworkers 19 co-cultured an intestinal epithelial cell line with Clostridium difficile on a polyethylene terephthalate electrospun structure to in vitro evaluate bacterial adhesion and the subsequent tissue infection. Thus, the use of a fibrous scaffold appeared promising being able to mimic the mechanical and physical properties of the native colonized tissue 20 .…”