Biofilm formation by Clostridium difficile

Abstract

Clostridium difficile infection (CDI) is a major healthcare-associated disease worldwide. Recurring infections and increasing antibiotic resistance have complicated treatment of CDI. While C. difficile spores are important for transmission and persistence of CDI, other factors such as gut colonization and formation of bacterial communities in the gut may also contribute to pathogenesis and persistence, but have not been well investigated. Recently, we reported that important clinical C. difficile strains are able to form composite biofilms in vitro. C. difficile biofilm formation is a complex process, modulated by several different factors, including cell surface components and regulators. We also reported that bacteria within biofilms are more resistant to high concentrations of vancomycin, the antibiotic of choice for treatment of CDI. Here we summarize our recent findings and discuss the implications of biofilm formation by this anaerobic gut pathogen in disease pathogenesis and treatment.

Keywords: Clostridium difficile; anaerobic pathogen; antibiotic resistance; biofilm formation; sporulation.

Figure 1. C. difficile biofilm formation in vitro. (A) Confocal microscopy analysis of biofilms formed by C. difficile R20291. Live/Dead staining shows dead bacteria red and live green, (propidium iodide and Syto 9, respectively). Biofilm was incubated for 3 days. 3D images of biofilms (right panel) depicting biofilm thickness in micrometers. Time course for biofilm formation by strain 630 (B) and R20291 (C) measured by CV staining (bars) and colony counts (CFU/ml, line). The results are presented in log scale, and the error bars represent standard deviations (P < 0.05).The data are representative of at least three independent experiments, each performed in triplicates. (D) Characterization of C. difficile biofilm matrix: 3D confocal microscopy images of R20291 biofilms stained with murine anti-R20291 after incubation for 3 days (E) Biofilms stained with antibodies to a synthetic C. difficile PSII polysaccharide (red) and DAPI (blue), which stains the bacterial DNA.

Figure 2. A hypothetical model for C. difficile biofilm development. Bacteria first recognize specific or nonspecific attachment sites on an appropriate surface and adhere to it. An intact S-layer and/or adhesins are important for this initial step of biofilm formation. The regulator of sporulation, Spo0A, controls this step, likely by upregulating expression of adhesins. Quorum sensing mediated by luxS is crucial during the early and late maturation phases when cells start to produce a biofilm matrix composed of proteins, extracellular DNA and polysaccharides. C. difficile flagella have a role in maturation of biofilms and perhaps in the early steps of biofilm formation, in directing bacteria to the right attachment sites. Spores are part of C. difficile biofilms, although numbers of spores may be maximal in mature biofilms where conditions of nutrient stress are likely. C. difficile biofilms can protect bacteria within from the antibiotic vancomycin, while at low concentrations (sub-inhibitory and inhibitory concentrations) biofilm formation is induced.