Fig 1.
Design of the CDC biofilm reactor and modified holder.
(A) Schematic of a general setup of a CDC biofilm reactor for biofilm growth. Source: Biosurface Technologies. (B) Components of a CDC biofilm reactor. (C) Customized rods into which collagen plugs were placed for biofilm growth. Labels detail modifications.
Fig 2.
Representative SEM images of collagen and polycarbonate coupon surfaces.
(A-C) Surface of a collagen coupon after soaking in broth only (no bacteria present). Deep valleys and ravines were consistent throughout the amorphous structure. (D-F) Surface of a polycarbonate coupon after soaking in broth only (no bacteria present). Ridges and plateaus had an undulating, yet mostly smooth surface, in particular relative to collagen.
Fig 3.
Representative SEM images of P. aeruginosa ATCC 27853 biofilm formation on both material types.
(A-C) Surface of a collagen coupon that had biofilm coverage. Biofilm structure conformed to the polymeric collagen network. As noted in Fig 2, deep valleys and ravines were consistent throughout the amorphous structure. (D-F) Surface of a polycarbonate coupon on which biofilms of P. aeruginosa ATCC 27853 grew. Biofilm structure was estimated to be greater than 20 cell layers thick. Growth followed the contour of the surface, even displaying the coupon machine marks (100x). Arrow (panel F) indicates extracellular matrix components that were observed.
Fig 4.
Representative SEM images of MRSA USA 400 biofilm on both material types.
(A-C) Surface of collagen with biofilm growth. Biofilm structure resulted in uniform coverage, but did not appear to plume as other staphylococcal isolates did. Rather, MRSA USA 400 displayed sheet-like growth on collagen. (D-F) Surface of a polycarbonate coupon that had sparse coverage, although where biofilm did form, it plumed to an estimated 30 cell layers thick. Arrow (panel F) indicates a biofilm plume.
Fig 5.
Representative SEM images of S. mutans ATCC 25175 biofilm formation on both material types.
(A-C) Surface of collagen with biofilm coverage. Cells of S. mutans filled the ravines and crevices of the collagen material. (D-F) In contrast to collagen, S. mutans grew in small plumes on the surface of polycarbonate with sporadic coverage. Arrow (panel E) indicates a biofilm plume.
Fig 6.
Representative SEM images of collagen coupons with three isolates pre- and post-vortex/sonication to demonstrate the ability of the process to remove bioburden from material surfaces.
(A-C) Representative images of biofilm structures prior to vortex and sonication. (D-F) Images of residual cells on collagen after vortex and sonication. Data indicated that biofilms were effectively disrupted/removed with <5% of cells remaining on the surface.
Fig 7.
Representative SEM images of S. epidermidis ATCC 35984 biofilms on collagen and polycarbonate coupons pre- and post-vortex/sonication.
(A) Heavy amounts of biofilm formed on collagen coupons, making the surface morphology unobservable. (B-C) Images showed that the large majority of biofilm burden had been removed by vortex/sonication, but clusters of cells still remained. (D) Similar to collagen, large plumes of biofilms formed on polycarbonate coupons with deep ravines. Although these ravines may have formed during the dehydration procedure, it is likely they were sites of water channels that provided fracture points within the biofilm communities. (E-F) Images showed that there was still a fair amount of surface coverage by biofilms post-vortex/sonication. However, it was estimated that there were <5% of cells remaining on the surface similar to other bacterial isolates examined.
Table 1.
Baseline (control) quantifications of coupons.
Table 2.
Bacteria and the antibiotics they were tested against, including MIC for each.
Table 3.
Average of Log10 transformed CFU/sample for each species and material at each concentration of antibiotic tested.
Unless otherwise noted (at times coupons fell out of the rod in the reactor and were not included in analysis), n = 6 collagen coupons were analyzed and n = 5 polycarbonate coupons were analyzed. It was hypothesized that biofilms of varying species grown on a complex collagen network would be less susceptible to antibiotics as compared to biofilms grown on polycarbonate coupons. The criteria for support of the hypothesis was a difference of 1 log10 unit or greater in bacterial counts between coupon types.