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. 2016 Oct 1:43:240-250.
doi: 10.1016/j.actbio.2016.07.016. Epub 2016 Jul 12.

Antifungal activity of a β-peptide in synthetic urine media: Toward materials-based approaches to reducing catheter-associated urinary tract fungal infections

Namrata Raman  1 , Myung-Ryul Lee  1 , Angélica de L Rodríguez López  2 , Sean P Palecek  3 , David M Lynn  4
Affiliations

Antifungal activity of a β-peptide in synthetic urine media: Toward materials-based approaches to reducing catheter-associated urinary tract fungal infections

Namrata Raman et al. Acta Biomater. .

Abstract

Catheter-associated urinary tract infections (CAUTI) are the most common type of hospital-acquired infection, with more than 30 million catheters placed annually in the US and a 10-30% incidence of infection. Candida albicans forms fungal biofilms on the surfaces of urinary catheters and is the leading cause of fungal urinary tract infections. As a step toward new strategies that could prevent or reduce the occurrence of C. albicans-based CAUTI, we investigated the ability of antifungal β-peptide-based mimetics of antimicrobial peptides (AMPs) to kill C. albicans and prevent biofilm formation in synthetic urine. Many α-peptide-based AMPs exhibit antifungal activities, but are unstable in high ionic strength media and are easily degraded by proteases-features that limit their use in urinary catheter applications. Here, we demonstrate that β-peptides designed to mimic the amphiphilic helical structures of AMPs retain 100% of their structural stability and exhibit antifungal and anti-biofilm activity against C. albicans in a synthetic medium that mimics the composition of urine. We demonstrate further that these agents can be loaded into and released from polymer-based multilayer coatings applied to polyurethane, polyethylene, and silicone tubing commonly used as urinary catheters. Our results reveal catheters coated with β-peptide-loaded multilayers to kill planktonic fungal cells for up to 21days of intermittent challenges with C. albicans and prevent biofilm formation on catheter walls for at least 48h. These new materials and approaches could lead to advances that reduce the occurrence of fungal CAUTI.

Statement of significance: Catheter-associated urinary tract infections are the most common type of hospital-acquired infection. The human pathogen Candida albicans is the leading cause of fungal urinary tract infections, and forms difficult to remove 'biofilms' on the surfaces of urinary catheters. We investigated synthetic β-peptide mimics of natural antimicrobial peptides as an approach to kill C. albicans and prevent biofilm formation in media that mimics the composition of urine. Our results reveal these mimics to retain structural stability and activity against C. albicans in synthetic urine. We also report polymer-based approaches to the local release of these agents within urinary catheter tubes. With further development, these materials-based approaches could lead to advances that reduce the occurrence of fungal urinary tract infections.

Keywords: Antifungal; Biofilms; Coatings; Multilayers; Urinary catheters; β-Peptides.

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Figures

Figure 1
Figure 1
(A) Circular dichroism spectra of β-peptide 1 in PBS (solid line), in minimal SU medium (dotted line), and in methanol (dashed line). (B) Planktonic antifungal activity of β-peptide 1 (solid circles) and antimicrobial α-peptide magainin-2 (open circles) in SU media. C. albicans cells (103 cells/mL) were incubated in the presence of 2-fold dilutions of peptide for 48 hours and XTT was used to assess metabolic activity. Data points are averages of at least two independent experiments with triplicates in each and error bars denote standard deviation. Arrow indicates MIC of the peptide in SU medium.
Figure 2
Figure 2
Fluorescence micrographs of C. albicans treated with β-peptide 1. Cells (105 cells/mL) were treated with the labeled β-peptide (yellow) at 2-fold varying concentrations from 0 to 64 µg/mL for 3.5 hours. Cells were stained with PI (red) to identify dead cells. Scale bars = 50 μm.
Figure 3
Figure 3
Fluorescence micrographs of catheter tube segments of polyethylene (A,D,G), polyurethane (B,E,H), and silicone (C,F,I) coated with multilayer films composed of PGA/PLL (A–C) or HA/CH (D–F) and incubated with β-peptide 1 for 24 hours. (G–I) Controls consisting of tube segments not coated with any multilayer film and incubated with β-peptide 1. Scale bars = 250 μm.
Figure 4
Figure 4
Plots of the release of β-peptide 1 into intraluminal buffer solution in polyethylene (circles), polyurethane (squares), and silicone (triangles) catheter tubes coated with β-peptide-loaded (A) PGA/PLL multilayers, (B) HA/CH multilayers, and (C) no multilayer film. Data points are averages of three technical replicates and error bars denote standard deviations.
Figure 5
Figure 5
Scanning electron micrographs of longitudinal sections of polyethylene (A,D,G,J), polyurethane (B,E,H,K), and silicone (C,F,I,L) catheter tube segments coated with (A–F) PGA/PLL and (G–L) HA/CH multilayers. The dotted lines indicate where PGA/PLL coated catheters were intentionally scratched to more clearly evaluate the presence of the coatings. Triangles indicate catheter walls, asterisks (*) indicate uniform multilayer coatings on the inner surface of the tube, and pound symbols (#) indicate areas of patchy multilayer coatings. Scale bars = 200 μm (A–C,G–I), 20 μm (D–F,J–L).
Figure 6
Figure 6
Antifungal activity of polyethylene catheter tubes coated with (A) PGA/PLL or (B) HA/CH multilayers loaded with antifungal β-peptide 1 (tube/film/pep) against C. albicans (106 cells/mL) in SU media. Antifungal activities of control untreated tube segments (tube) and tubes coated with multilayers but not loaded with β-peptide (tube/film) are also shown. Data points are averages of three independent experiments consisting of three technical repeats each and error bars denote standard deviation. Colony counts of solutions from tubes coated with β-peptide-loaded films (tube/film/pep) were statistically different (p < 0.05 by two-tailed t-test) from colony counts of solutions from untreated controls (tube).
Figure 7
Figure 7
Antifungal activity of polyethylene catheter tubes coated with (A) PGA/PLL and (B) HA/CH in SU media after pre-incubation with PBS for extended times. Catheter tubes coated with multilayer film and loaded with β-peptide (black bars), tubes coated with multilayer films (no peptide; grey bars), and untreated bare control tubes were filled with PBS for pre-determined times, as indicated on the x-axis, after which the buffer was removed and antifungal activity was evaluated by adding C. albicans inoculum and using XTT to evaluate metabolic activity. Data points shown are average of three replicates and all values are normalized to the metabolic activity of the untreated catheter tubes at the same time point. For all pre-incubation times, reductions in metabolic activity for β-peptide-loaded films (tube/film/pep) were statistically different (p < 0.05 by two-tailed t-test) from untreated controls (tube) under the same conditions.
Figure 8
Figure 8
Lower (A–E) and higher (F–J) magnification scanning electron micrographs of C. albicans biofilms in SU media on (A,F) untreated polyethylene catheter tubes, (B,G) tubes coated with PGA/PLL multilayers, or (C,H) HA/CH multilayers, and (D,I) PGA/PLL films and, (E,J) HA/CH films loaded with β-peptide. The dotted boxes in A–E indicate the approximate region of the catheter from which the higher magnification images in F–J were taken. Biofilms were grown on the inner surfaces of polyethylene catheter tube segments in SU media for 48 hours and tubes were longitudinally sliced open and prepared for imaging. Scale bars = 200 μm (A–E), 20 μm (F–J).
Figure 9
Figure 9
Quantitative characterization of biofilm formed in polyethylene (PE, black), polyurethane (PU, grey), and silicone (white) catheter tubes coated with (A) PGA/PLL and (B) HA/CH multilayer films loaded with β-peptide (tube/film/pep). Controls consisting of catheter tubes coated with PGA/PLL or HA/CH multilayer films (no peptide; tube/film) and untreated bare catheter tubes (tube) are also indicated. C. albicans biofilms were grown in the tubes for 48 hours in SU medium, after which an XTT assay was performed to quantify the metabolic activity of the biofilms. Data points represent the average of three replicates from three independent experiments each and error bars denote standard deviation. For all catheter tube material, metabolic activity observed from β-peptide-loaded films (tube/film/pep) were statistically different (p < 0.05 by two-tailed t-test) from untreated controls (tube) under the same conditions.
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