A surfactant polymer dressing potentiates antimicrobial efficacy in biofilm disruption

Abstract

A 100% water-soluble surfactant polymer dressing (SPD) that is bio-compatible and non-ionic has been reported to improve wound closure in preliminary clinical studies. The mechanism of action of SPD in wound healing remains unclear. Biofilm infection is a significant problem that hinders proper wound closure. The objective of this study was to characterize the mechanism of action of SPD inhibition of bacterial biofilm development. Static biofilms (48 h) of the primary wound pathogens Pseudomonas aeruginosa (PA01), Staphylococcus aureus (USA300) were grown on polycarbonate membranes and treated with SPD with and without antibiotics for an additional 24 h. The standard antibiotics - tobramycin (10 μg/ml) for PA01 and rifampicin (10 μg/ml) for USA300, were used in these studies. Following 24 h treatment with and without antibiotics, the biofilms were characterized using scanning electron microscopy (SEM) structural imaging, in vitro imaging system (IVIS) proliferation imaging, colony forming units (CFU), viability assay, quantitative PCR (qPCR) for virulence gene expression. Because SPD is a surfactant based dressing, it potentially has a direct effect on Gram negative bacteria such as Pseudomonas primarily due to the lipid-based outer membrane of the bacteria. SPD is a surfactant based dressing that has potent anti-biofilm properties directly or in synergy with antibiotics.

Figure 1

SPD inhibits Rhl regulated pyocyanin production by P. aeruginosa PA01. (A) Biofilm co-aggregation observed in the no treatment PA01 culture was not observed in SPD treated 48-72 hours cultures, n = 6. (B) Bar graph showing mean levels of pyocyanin in control and SPD treated samples. Data are shown mean ± SD, n = 6, *p < 0.05. (C) The total ion signal chromatograms of pyocyanin and internal standard norharmane produced by P. aeruginosa PA01 in normal condition (con) and in the presence of compound (SPD). The retention times for pyocyanin and norharmane with the solvent system used in this study were 8.1 min and 9.25 min, respectively. The total ion signal chromatograms of pyocyanin and norharmane (internal standard) produced by P. aeruginosa PA01 in normal condition (con) and in the presence of compound (SPD). The retention times for pyocyanin and norharmane with the solvent system used in this study were 8.1 min and 9.25 min, respectively. (D) P aeruginosa PA01 was either left untreated or treated for 24 h with SPD. mRNA was isolated and real-time PCR was performed to assess rhlR gene expression in PA01.

Figure 2

SPD disrupts biofilm matrix in P. aeruginosa PA01 and S. aureus USA300. Triple staining with DAPI (DNA), SYPRO Ruby (biofilm matrices) and FITC-Wheat Germ Agglutinin (membrane glycoproteins) was performed for P. aeruginosa PA01. (A) Shown are representative images of PA01 biofilms that were untreated or SPD treated and stained with the respective dyes. Graphical representation of staining intensity for (C) SYPRO Ruby and (D) WGA are shown. Data are shown mean ± SD, n = 6. *p≤0.05. Scale bar = 40 µM. Triple staining with DAPI (DNA), SYPRO Ruby (biofilm matrices) and FITC-Wheat Germ Agglutinin (membrane glycoproteins) was performed for S. aureus USA300. (B) Shown are representative images of USA300 biofilms that were untreated or SPD treated and stained with the respective dyes. Graphical representation of staining intensity for (E) SYPRO Ruby and (F) WGA are shown. Data are shown mean ± SD, n = 6. *p≤0.05. Scale bar = 40 µM.

Figure 3

SPD decreases metabolic activity of bacteria. Shown are representative IVIS images and corresponding graphical representations of intensity quantitation from 48 h biofilms of (A,B) P. aeruginosa Xen41 and (C,D) S. aureus SAP231 either left untreated, or treated for 24 h with antibiotic (tobramycin (tobra) or rifampicin (rifam) respectively) alone, SPD alone or a combination of SPD and antibiotic. Data are shown mean ± SD, n = 8, *p < 0.05. Also included is a heat map showing intensity ranges. The intensity of blue and red signals were quantified using ImageJ and shows that bacteria in both control and antibiotic treated group show higher metabolic activity whereas SPD and combination therapy resulted in decreased metabolic activity.

Figure 4

SPD acts synergistically with antibiotics and disrupts biofilm structural integrity. 48 h static biofilms of (A) P. aeruginosa PA01 and (B) S. aureus USA300 were either left untreated or treated for 24 h with SPD or antibiotic (tobramycin (tobra) or rifampicin (rifam)) alone or in combination. Shown are representative scanning electron micrographs (SEM) at 2500× and 10000× magnifications. SPD alone or in combination with antibiotics showed fewer dense aggregates of bacteria with associated matrix. n = 6. Scale bar = 10 μm, 2500× magnification and 5 μm, 10000× magnification.