Preventing Pseudomonas aeruginosa Biofilms on Indwelling Catheters by Surface-Bound Enzymes

Abstract

Implanted medical devices such as central venous catheters are highly susceptible to microbial colonization and biofilm formation and are a major risk factor for nosocomial infections. The opportunistic pathogen Pseudomonas aeruginosa uses exopolysaccharides, such as Psl, for both initial surface attachment and biofilm formation. We have previously shown that chemically immobilizing the Psl-specific glycoside hydrolase, PslG_h, to a material surface can inhibit P. aeruginosa biofilm formation. Herein, we show that PslG_h can be uniformly immobilized on the lumen surface of medical-grade, commercial polyethylene, polyurethane, and polydimethylsiloxane (silicone) catheter tubing. We confirmed that the surface-bound PslG_h was uniformly distributed along the catheter length and remained active even after storage for 30 days at 4 °C. P. aeruginosa colonization and biofilm formation under dynamic flow culture conditions in vitro showed a 3-log reduction in the number of bacteria during the first 11 days, and a 2-log reduction by day 14 for PslG_h-modified PE-100 catheters, compared to untreated catheter controls. In an in vivo rat infection model, PslG_h-modified PE-100 catheters showed a ∼1.5-log reduction in the colonization of the clinical P. aeruginosa ATCC 27853 strain after 24 h. These results demonstrate the robust ability of surface-bound glycoside hydrolase enzymes to inhibit biofilm formation and their potential to reduce rates of device-associated infections.

Keywords: Pseudomonas aeruginosa; PslGh; bacterial biofilms; biomaterials; catheters; enzyme immobilization; glycoside hydrolases; medical device infection.

Figure 1.

(A) Schematic illustration of the sequence of steps involved in enzyme immobilization: surface activation by plasma treatment; amine (NH2) functionalization with 3-aminopropyltrimethoxysilane (APTMS) via silanization; activation of the catheter lumen surface using the bifunctional agent (GDA) under the formation of Schiff base and finally coupling of the enzyme. (B) Chemical immobilization of PslG_h prevents P. aeruginosa biofilm formation on the luminal surface of catheter tubing. Schematic representation demonstrating cell attachment and biofilm formation on untreated catheters (top) and inhibition of biofilm formation when the catheter is treated with PslG_h (bottom).

Figure 2.

Chemical and physical characterization of the lumen of commercial polymer tubing and a medical grade catheter. (A-C) ATR-FTIR spectra showing the surface chemical groups lining the PE, PU and PDMS tubing lumen during immobilization; (D) Variation in the relative water level inside PE tubing after each immobilization step. A rise in the water level within catheter lumen is indicative of hydrophilic character; and (E) Relative displacement of water inside 4 lumen segments (S1-S4) of a 10cm long PE-100 catheter after treatment with atmospheric plasma for 10 min. ****P ≤ 0.0001. NS, no significant difference.

Figure 3.

Bound PslG_h prevents P. aeruginosa biofilm formation on the luminal surface of polymer tubing. (A) Florescence images showing the inhibition of biofilm formation by bound PslG_h on the luminal surface of PE, PU and PDMS tubing, but not for the untreated control. (B) Corresponding cell counts (per cm²) calculated from the image analysis of surfaces in (A). ****P ≤ 0.0001.

Figure 4.

PslG_h-modified PE-100 catheter inhibits biofilm formation (A) Effective concentration of immobilized PslG_h required for biofilm inhibition. Effect of PslG_h concentration (μg/mL) in immobilization solution on its binding capacity to catheter luminal surface (μg/cm) (left). Effect of immobilized PslG_h surface density on biofilm formation of Pseudomonas aeruginosa PAO1 (right). (B) Anti-biofilm activity of the PE catheter lumen with bound PslG_h, relative to the untreated control, or APTMS and GDA functionalized surfaces. (A) Colony forming units per cm² (CFU) (top), corresponding florescence images (middle), and crystal violet (CV) stained images (bottom). All measurements were acquired after incubation for 24 h in bacterial culture. Biofilms were stained with SYTOX Green (middle) and CV (bottom) (scale bar, 50 μm). ****P ≤ 0.001. NS, no significant difference.

Figure 5.

Antibiofilm activity of the lumen of high density PslG_h-bound 10 cm PE-100 catheters against P. aeruginosa. (A) Photos of CV stained 10 cm PE-100 catheters; (B) Fluorescence images of 5 alternating segments (i.e., S2, 4, 6, 8 and 10); and (C) Colony forming units per cm² (CFU) of the other 5 alternating segments (i.e., S1, 3, 5, 7 and 9). All measurements were acquired after incubation for 24 h in bacterial culture. Biofilms were stained with CV in (A) and with SYTOX Green in (B) (scale bar, 50 μm). *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001.

Figure 6.

Inhibition of P. aeruginosa biofilm formation by PslG_h bound to the lumen surface of PE-100 catheter under dynamic flow culture conditions. (A) Fluorescence images of biofilm growth for up to 14 days on the lumen surface of untreated (top) and PslG_h-bound PE-100 catheter (bottom). (B) Colony forming units (CFU/cm²) of P. aeruginosa PAO1 on the lumen surface of untreated and PslG_h-bound PE-100 catheter after 14 d incubation. Biofilms were stained with SYTOX Green in (A) (scale bar, 100 μm). ****P ≤ 0.0001.

Figure 7.

In vivo inhibition of biofilm formation of P. aeruginosa ATCC 27853 on the PslG_h-treated lumen surface of PE catheter. (A) SEM images of biofilm growth on untreated and treated PE-100 catheters at low magnification (1,000X), and (B) corresponding bacterial burden showing ~ 2 log CFU /cm² reduction in P. aeruginosa ATCC 27853 cells after 1d of placement in vivo. The scale bars represent 20 μm, respectively. ****P ≤ 0.0001.