(A-4) Time-dependence of Staphylococcus epidermidis Biofilm Dispersal and Dispersed Cell Adhesion to Protein-Treated Surfaces

Sub-track: Other/Non-specified

Bacterial biofilms are aggregates of bacteria surrounded by self-produced viscoelastic extracellular polymeric substances. Bacterial aggregation into biofilms presents one of the most effective methods used by pathogenic bacteria to mitigate the activity of antimicrobial agents and enable bacterial infections to spread via the dispersal of cells from biofilms. Staphylococcus epidermidis is the most common bacteria found on human skin and is ordinarily a commensal. However, it becomes pathogenic and often forms biofilms when contaminating medical devices or implants during insertion. S. epidermidis biofilms can then spread and adhere to secondary sites in other parts of the body, exacerbating infection. The material properties and concentration of dispersed cells are important to the spread of infection.  This study characterizes the time-dependent dispersion of cells from biofilms, as well as the subsequent adhesion of dispersed cells to physiologically relevant protein-coated surfaces (fibronectin and collagen).  A deeper understanding of the time-dependence of bacterial cell dispersal and dispersed cell adhesion will enable the development of innovative biofilm control methods that account for the material properties and concentration of dispersed cells and dispersed cell clusters.

Staphylococcus epidermidis RP62A (ATCC 35984) biofilms were grown at a shear stress of 0.1 Pa in poly-dimethyl siloxane (PDMS) flow cells for up to 48 hours. Effluent containing bacteria dispersed from the biofilms was collected after 6, 12, 18, 24, and 48 hours. Effluent was collected for 1 hour in four 15-minute increments to minimize bacterial growth during collection.  Effluent was subsequently imaged in a hemocytometer with phase-contrast microscopy.  Bacterial concentration (cells/mL) was quantified using hemocytometer cell counts.  The collected effluent was also used to assess the adhesion of dispersed cells to glass, fibronectin-treated glass, and collagen-treated glass.  Briefly, the collected effluent was incubated on the untreated or treated glass substrate for 1 hour at 37°C and washed two times with phosphate-buffered saline (PBS). Samples were stained with fluorescent stain (SYTO-9) and adherent cells were imaged using confocal scanning laser microscopy (CLSM).  Differences in adhesion were assessed using image analysis across untreated and treated glass surfaces. 

The concentration of cells dispersed from biofilms was highest after 18 hours of biofilm development.  The concentration of dispersed cells released from the biofilms increased from 6 to 18 hours, then decreased slightly from 18 to 24 hours before remaining relatively constant between 24 and 48 hours. This indicates that as biofilms mature, biofilm dispersion rates peak and then plateau to a relatively steady rate of cell dispersion.  Additionally, we report that dispersed S. epidermidis cells adhere in higher concentrations across glass, fibronectin, and collagen surfaces compared to planktonic cells due to increased clustering of dispersed cells.  This research advances the fundamental understanding of the biofilm lifecycle by evaluating links between biofilm maturation, bacterial biofilm dispersal, and dispersed cell adhesion to physiologically relevant protein-coated surfaces.   

S. epidermidis biofilm dispersal is found to fluctuate during biofilm development from 6 to 48 hours with a peak concentration of dispersed cells after 18 hours of biofilm growth.  Additionally, we show that dispersed cells adhere to untreated and protein-treated substrates in higher concentrations than planktonic bacteria. The adhesion rates for dispersed biofilm cells emphasize the significance of cells dispersed from biofilms in secondary infections. Characterizing the dispersal and adhesion of cells released from S. epidermidis biofilms will inform the development of antimicrobial treatments and antifouling biomaterials that account for fluctuations in bacterial biofilm dispersal and the adhesive properties of dispersed cells. This knowledge will ultimately contribute to reductions in nosocomial infection rates and the burden of S. epidermidis infections on healthcare systems.