Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile

Abstract

Bacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

Fig 1

C. difficile biofilm formation in vitro. (A) Biofilm formation by strain 630 and hypervirulent strain R20291, in BHIS supplemented with 0.1 M glucose or 0.3 M NaCl, for 3 days at 37°C in anaerobic conditions. Biofilm formation was measured by CV staining. (B and C) Time course for biofilm formation for strains 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 and E) Photographs of biofilms formed on a 24-well plate for strains 630 and R20291 on day 1 (D) and day 3 (E) are shown.

Fig 2

Confocal microscopy analysis of biofilms formed by C. difficile. Live/Dead staining shows dead (red) and live (green) bacteria (propidium iodide and Syto 9, respectively) in strains R20291 (A and B) and 630 (C and D) biofilms after incubation for 1 day (A and C) and 3 days (B and D). 3D images of biofilms depicting biofilm thickness in μm are shown in the left panels.

Fig 3

Characterization of C. difficile biofilm matrix. (A) shows 3D confocal microscopy images of staining of R20291 biofilms with murine anti-R20291 (left panel) and mouse preimmune serum (right panel) after 3 days of incubation. (B) Biofilms stained with Ruby matrix stain after 3 days of incubation. Biofilms were stained with antibodies to a synthetic C. difficile PSII polysaccharide (red) and DAPI (blue), which stains the bacterial DNA (C), or with the control mice preimmune serum and DAPI (D). Biofilms were incubated with proteinase K (E) or DNase I (F) as described in Materials and Methods. The dark gray bars represent data from treatment of either enzyme at the start of incubation (inhibition of biofilm formation), and the light gray bars represent data from incubating preformed 1-day-old biofilms with either enzyme (disruption of biofilms). The data shown are representative of at least two independent experiments performed in triplicates (P < 0.05).

Fig 4

Role of S layer and flagella in biofilm formation. (A) Biofilm formation by WT R20291, a cwp84 mutant (Δcwp84) for days 1, 3, and 5 and a complemented strains (cwp84-C) for day 1 in vitro as measured by CV staining. (B) Biofilm formation by WT R20291, fliC mutant (fliC) for days 1, 3, and 5 and complemented strains fliC (fliC-C) for day 5 in vitro as measured by CV staining. (C) Confocal microscopy images of Live/Dead staining of biofilms formed by the WT, Δcwp84, cwp84-C, and fliC. The results are presented in log scale, and the error bars represent standard deviations (P < 0.05). Biofilm assays were performed in triplicates, and data are representative of at least three independent experiments.

Fig 5

Potential role for quorum sensing in C. difficile biofilm formation. (A) Biofilm formation by WT R20291, putative quorum-sensing gene luxS mutant (luxS), and complemented strains (luxS-C) as measured by CV staining. (B) Confocal microscopy analysis of WT, luxS, and complemented strain luxS-C. The results are presented in log scale, and the error bars represent the standard deviations (P < 0.05). The data from biofilm assays are representative of at least three independent experiments performed in triplicates.

Fig 6

Sporulation/germination proteins affect C. difficile biofilm formation. (A) Quantitation of the number of spores present in the adherent, planktonic phases of biofilm and in planktonic tube culture, in brain heart infusion media (BHI) with sodium taurocholate (BHI+T) and heat treatment (80°C), as described in Materials and Methods. (B and C) Biofilm formation by WT R20291, sporulation transcription factor spo0A mutant (spo0A) and complemented spo0A mutant (spo0A-C) after 1 day (B) and WT R20291, sleC mutant (sleC) and complemented sleC mutant (sleC-C) after 3 days (C). (D) Confocal microscopy analysis of WT and mutants spo0A, complemented spo0A mutant (spo0A-C), and sleC. The results are presented in log scale, and the error bars represent standard deviations (P < 0.05). Both biofilm and spore quantitation experiments were performed in triplicates, and the data shown are representative of at least three independent experiments.

Fig 7

Effect of antibiotics on C. difficile biofilms. (A) Clostridium difficile 1- to 3-day-old biofilms and the corresponding planktonic growth were exposed to 20 μg of vancomycin/ml (100 times the MIC) for 24 h. The data are presented as percentage of surviving bacteria after treatment with antibiotics for 24 h for 1- and 3-day-old biofilms. (B) Bacterial counts from disrupted 1-day-old biofilms were incubated with 20 μg of vancomycin/ml for 6 h or 24 h. (C) Biofilm formation measured by CV staining at days 1 and 3 after treatment with subinhibitory and inhibitory concentration of antibiotic vancomycin (MIC for R20291 was 0.2 μg/ml). The results are presented in log scale, and the error bars represent standard deviations (P < 0.05). An asterisk (*) denotes significant differences compared to biofilm formation in the absence of vancomycin (0 μg/ml).