Wolf in Sheep's Clothing: Clostridioides difficile Biofilm as a Reservoir for Recurrent Infections

Abstract

The microbiota inhabiting the intestinal tract provide several critical functions to its host. Microorganisms found at the mucosal layer form organized three-dimensional structures which are considered to be biofilms. Their development and functions are influenced by host factors, host-microbe interactions, and microbe-microbe interactions. These structures can dictate the health of their host by strengthening the natural defenses of the gut epithelium or cause disease by exacerbating underlying conditions. Biofilm communities can also block the establishment of pathogens and prevent infectious diseases. Although these biofilms are important for colonization resistance, new data provide evidence that gut biofilms can act as a reservoir for pathogens such as Clostridioides difficile. In this review, we will look at the biofilms of the intestinal tract, their contribution to health and disease, and the factors influencing their formation. We will then focus on the factors contributing to biofilm formation in C. difficile, how these biofilms are formed, and their properties. In the last section, we will look at how the gut microbiota and the gut biofilm influence C. difficile biofilm formation, persistence, and transmission.

Keywords: CDI relapsing; Clostridioides difficile infection; biofilm inducers; colonisation resistance; commensal microbiota; dysbiosis; mucosal-biofilm; persistence.

Figure 1

Healthy microbiota biofilms versus a dysbiotic microbiota biofilms. In a healthy microbiota (left panel), the microbial density and diversity increase from the stomach to the colon. In the small intestine, biofilms are discontinuous and loose aggregates, while in the large intestine, biofilms are dense, continuous and attached to a uniform mucus layer (attached biofilms). The biofilms in the gut lumen are loosely attached to food particles or encapsulated in mucin (aggregate biofilms). Commensal biofilms facilitate the host barrier function by thickening the mucus layer, regulating the secretion of IgA, stimulating conversion of pro-IL-1β into active IL-1β and inducing the development of Th17 cells. A dysbiotic microbiota (right panel) presents (1) damaged mucus-biofilm exposing epithelium cells to luminal content or (2) invasive biofilms where bacteria come directly into contact with the epithelium. Both scenarios expose the intestinal epithelium to pathogens and pathobionts which can trigger an infection. Invasive polymicrobial biofilms could trigger cellular inflammation, abnormal cellular proliferation, increased epithelial permeability (activation of IL-6 and Stat3) in patients with colorectal cancer (CRC), increased IL-17 production and DNA damage in patients with familial adenomatous polyposis (FAP), and inflammatory bowel disease (IBD). Patients’ Adherent-invasive E. coli (AIEC) colonize the intestinal mucosa and stimulate the secretion of TNF-α and mucin degradation.

Figure 2

Model for C. difficile aggregate/attached biofilm formation in vitro. The first step toward biofilm formation is either attachment of the cells to a surface or cellular aggregation. In both cases a shift in surface structures controlled by rising c-di-GMP levels results in the replacement of the flagella by T4P and adhesins (collagen and fibronectin binding proteins). Autolysin-mediated cell lysis is likely the main mechanism contributing to the formation of the extracellular matrix by releasing chromosomal DNA and cellular proteins in the medium, and exopolysaccharides may be synthesized and contribute to the matrix. Quorum sensing may induce prophage lysis that would also contribute to the biofilm matrix. Furthermore, c-di-GMP levels remain relatively high, ensuring consistent T4P and adhesins expression. C. difficile biofilm formation is characterized by a metabolic shift from glycolysis and the pentose phosphate pathway to the Stickland fermentation pathways and the Wood-Ljungdhal pathway, which are less efficient at producing energy. The table summarizes information about the main mechanisms involved in biofilm formation. Up-regulated mechanisms are indicated by the red upward arrows and the down-regulated mechanisms are indicated by the blue downward arrows. Abbreviations: aa: amino acids; QS: quorum sensing; eDNA: extracellular DNA; EPS: exopolysaccharides; DOC: deoxycholate; FOS: fructooligosaccharides.

Figure 3

Proposed model for the persistence of C. difficile. In this model, spores (circles) and biofilms contribute to short term and long-term relapses, respectively. Spores encased in mucus, biofilm communities or engulfed by epithelial cells, would eventually be eliminated by the renewal of the mucus and epithelial cells. The vegetative cells (rods) would keep a small viable population resulting in a biofilm that would be resistant to renewal of the mucus layers and epithelial cells. Sporulation could occur in the deeper layers of the biofilm, keeping a continuous supply of spores and leading to long-term relapses.

Figure 4

Proposed life cycle of C. difficile based on the metabolic landscape of the gut. The metabolic landscape is determined by 3 variables: competition, availability of microbiota-derived nutrients and availability of host-derived nutrients. Concentration of antimicrobial compounds and the metabolic landscape will determine the C. difficile growth rate and toxin production. Specifically, biofilm-persistence is a response to ecological competition caused by restriction in nutrient availability due to moderate levels of competition from the resident microbiota. Pathogenesis is a response to nutritional stress caused by a decrease in the availability of nutrient due to overgrowth. Sporulation is a response to starvation due to the depletion of nutrients in the gut or localized in the deeper layer of the biofilm. +++ high, ++ Moderate, + low, +/− very low.