Biofilm Assays on Fibrinogen-coated Silicone Catheters and 96-well Polystyrene Plates

Abstract

Biofilm formation is a well-known bacterial strategy that protects cells from hostile environments. During infection, bacteria found in a biofilm community are less sensitive to antibiotics and to the immune response, often allowing them to colonize and persist in the host niche. Not surprisingly, biofilm formation on medical devices, such as urinary catheters, is a major problem in hospital settings. To be able to eliminate such biofilms, it is important to understand the key bacterial factors that contribute to their formation. A common practice in the lab setting is to study biofilms grown in laboratory media. However, these media do not fully reflect the host environment conditions, potentially masking relevant biological determinants. This is the case during urinary catheterization, where a key element for Enterococcus faecalis and Staphylococcus aureus colonization and biofilm formation is the release of fibrinogen (Fg) into the bladder and its deposition on the urinary catheter. To recapitulate bladder conditions during catheter-associated urinary tract infection (CAUTI), we have developed a fibrinogen-coated catheter and 96-well plate biofilm assay in urine. Notably, enterococcal biofilm factors identified in these in vitro assays proved to be important for biofilm formation in vivo in a mouse model of CAUTI. Thus, the method described herein can be used to uncover biofilm-promoting factors that are uniquely relevant in the host environment, and that can be exploited to develop new antibacterial therapies.

Keywords: Biofilm; CAUTI; Catheter; Enterococcus faecalis; Fibrinogen; Infection; Urine.

Figure 1.. Protocol layout

Figure 2.. Preparation of 1 cm silicone pieces.

A. Silicone tubing. B. 1 cm silicone pieces. C. 1 cm pieces in half.

Figure 3.. Representative data of biofilm measured by colony forming units (CFU).

Comparison of biofilm formation between E. faecalis OG1RF (wild-type strain) and a manganese uptake system triple mutant strain (ΔefaΔmntH1ΔmntH2), which is a biofilm deficient mutant. Two-tailed Mann-Whitney U tests were performed to determine significance between two groups (***p < 0.0002).

Figure 4.. Representative images and data of biofilm analyzed by immunostaining and crystal violet.

A. Visualization of biofilm formation on catheter pieces by E. faecalis OG1RF and a manganese uptake system triple mutant strain (ΔefaΔmntH1ΔmntH2). Catheter controls are those Fg-coated catheters that were incubated with only urine (no bacteria). B. Immunostaining analysis of biofilm formation between OG1RF WT and deficient mutant by plotting fluorescence relative to catheter controls. C. CV staining to quantify and compare biofilm formation between OG1RF WT and deficient mutant by plotting absorbance relative to catheter controls. Two-tailed Mann-Whitney U tests were performed to determine significance between two groups (***p < 0.0002).