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 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 ~1.5-log reduction in the colonization of the clinical P. aeruginosa ATCC 27853 strain after 24 hours. 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.

Graphical Abstract

1. Introduction

Biofilms formed on implanted medical devices are responsible for 50–70% of all hospital acquired infections, and can be tremendously difficult to treat ^1–4. Biofilms are surface-attached microbial colonies embedded within a self-produced extracellular matrix, composed of exopolysaccharide, extracellular DNA and proteins ⁵. Annually, there are an estimated 14 million biofilm infections in the US, causing at least 350,000 deaths ⁴. The vast majority of these infections (86–95%) are associated with microbial colonization of catheters e.g., central-line associated bloodstream infections (CLABSI), catheter-associated urinary tract infections (CAUTI), and ventilator-associated pneumonia (VAP) ^6–7. The annual cost of the top five hospital-acquired infections is ~$9.8 billion in the US alone ⁸. Indwelling central venous catheters (CVC) are routinely used to administer medications, blood products, and nutritional fluids to patients, for both temporary and long-term use⁹. The clean surfaces of newly-implanted catheters are highly susceptible to microbial colonization ^10–13. The exopolysaccharide component of the matrix impairs antibiotic penetration ^14–15 and provides a barrier against phagocytosis by host immune cells ¹⁶. Within biofilms, microbes are up to 1000 times more tolerant to antimicrobials than planktonic cells ^{1, 10, 17–18}. As a result, device-related biofilm infections are difficult to treat using systemic antibiotics ¹⁹, and often require device extraction and replacement.

Medical biofilms can involve a wide variety of organisms, but the bacterium Pseudomonas aeruginosa accounts for ~28% of device-related infections ^20–23. P. aeruginosa is an ubiquitous, Gram-negative, opportunistic pathogen associated with a wide array of life-threatening acute and chronic nosocomial infections, particularly in patients with compromised host-defense mechanisms ²⁴. P. aeruginosa infections are becoming more challenging to treat due to the emergence of multidrug resistant strains ^{14–15, 25–28}. P. aeruginosa produces three distinct biofilm exopolysaccharides: Psl, Pel, and alginate ^29–31. The cationic Pel and neutral Psl polysaccharides are produced in varying amounts in different strains ³², while the anionic alginate is synthesized exclusively by mucoid strains in the lungs of cystic fibrosis patients ^29–30. Both Pel and Psl play a significant role in the formation and maintenance of biofilm structure ^{14, 33}, and recent work has shown the role that polysaccharides (such as Psl) play to enable the cell-cell and cell-surface adhesion necessary for biofilm formation ^{14, 16, 32, 34–35}.

Antimicrobial coatings for medical devices often incorporate silver, chlorhexidine, or triclosan for diffusive release ^{13, 36–38}. These coatings are often ineffective over time as they do not directly prevent microbial attachment, and new cells eventually accumulate on dead cells ³⁹. Diffusional release is also limited by a number of factors, including: (i) slow diffusion rates to the material surface; (ii) decreased activity over time as the antimicrobial concentration decreases and bacterial biomass accumulates ⁴⁰; and (iii) antimicrobial tolerance ^41–42. These limitations have led to an alternative trend to develop non-adhesive, non-fouling surfaces that target the early stages of biofilm development. These materials are designed to keep bacteria in a planktonic (swimming) state ^43–45 and thus more susceptible to antibiotics and the host immune system ¹⁰. Non-fouling, ‘bio-passive’ polymer brush surfaces, such as hydrophilic polyethylene glycol (PEG) are effective, but tend to fail after a period of days ^46–47. Slippery liquid infused porous surfaces (SLIPS) show highly-effective, long term anti-fouling properties but clinical performance is still unclear ⁴⁸.

Recently, we developed novel ‘bioactive’ biomaterial surfaces by covalently immobilizing the glycoside hydrolase, PslG_h. In solution, this enzyme has been shown to disrupt P. aeruginosa biofilms in vitro ^49–50. We immobilized PslG_h to flat squares of glass, polydimethylsiloxane (PDMS) and polystyrene (PS), and demonstrated that the surfaces prevented adsorption by hydrolyzing Psl ⁵¹. The PslG_h-bound surfaces reduced P. aeruginosa biofilms by 3–4 log units (8 d in static culture), compared to controls. However, to be suitable for clinical application, it is important to uniformly immobilize PslG_h along the length of the (inner) lumen surface of medical-grade catheters, and demonstrate long term activity to prevent P. aeruginosa biofilm growth. Herein, we show that PslG_h surface-modified catheters of polyethylene (PE), PDMS and polyurethane (PU) not only exhibit significantly reduced rates of biofilm formation under flow culture in vitro but also in vivo using a rat catheter infection model.

2. Materials and Methods

2.1. Materials

Aminopropyl trimethoxysilane (APTMS), glutaraldehyde (GDA), ethanol (HPLC grade), Fetal bovine serum (FBS) and all other reagents were of high analytical grade and were purchased from Sigma Aldrich (Mississauga, ON). Commercial polyethylene (PE), polyurethane (PUR) and polydimethylsiloxane silicone (PDMS) tubing with identical dimensions (0.3 cm ID and 0.6 cm OD) from McMaster-Carr (Ohio, USA), and medical grade polyethylene (PE-100) catheters (0.8 mm, ID) (Intramedic, Becton Dickinson and Company, NJ) were used as biomaterial surfaces.

2.2. Strains, media and growth conditions

Strains used in this study are P. aeruginosa PAO1 (wild-type strain; serotype O5) and P. aeruginosa ATCC 27853 (clinical strain). Lysogeny broth (LB) and agar were used for standard culture conditions. To prepare a pre-culture of tested strains, a single colony of freshly grown bacterial cells grown on LB agar overnight at 37 °C was inoculated in 5 mL LB and incubated at 37 °C overnight with agitation. Bacterial biofilms were grown in LB without salt (LBNS). Serum treatment was performed by coating the catheter segment with 100 μL of FBS then incubating without shaking at room temperature for 30 min.

2.3. Enzyme purification

The hydrolase domain of PslG_h was cloned into an expression plasmid and purified from E. coli as previously described ^{50, 52}. Briefly, the enzyme was recombinantly expressed in E. coli cells and grown in autoinduction media overnight at 37 °C with 50 μg/mL kanamycin. The protein was purified by Ni-NTA affinity chromatography using an N-terminal His₆-tag present on the enzyme followed by size exclusion chromatography (HiLoad 16/600 Superdex 200 pg, GE Healthcare). The protein was eluted in 20 mM Tris (pH 7.5), 150 mM NaCl, and 2% (vol/vol) glycerol and stored at −80 °C until required. The His₆-tag was left intact for all experiments.

2.4. Surface modification and enzyme immobilization

Enzyme immobilization by covalent binding for the activated surfaces was performed as previously described ⁵¹. We took advantage of surface hydroxyl or carboxylic groups as reactive sites for the APTMS, creating primary amines to be covalently linked to those of the PslG_h by GDA (Figure 1A). All polymer sheets and tubing (PE, PU and PDMS) and medical grade PE catheters (PE-100) were sonicated in absolute ethanol for 15 min then oven dried. Dried samples were exposed to atmospheric plasma (Harrick Plasma PDC001) at high power for up to 10 min. To ensure an even hydroxylation of the lumen surface, sections were flipped end to end after 5 min then exposed to plasma for additional 5 min. Samples were then functionalized with amine (NH₂) groups by immersion in APTMS (Sigma-Aldrich, 97%) solution (50 mg/mL in 80% (v/v) ethanol) for 2 h with shaking at 25 °C according to a published protocol ⁵³. Tubing segments were checked for any air bubbles that may prevent the APTMS contact with the lumen. After silanization, the surfaces were washed three times with 80% (v/v) ethanol to remove unreacted APTMS. The amino-functionalized surfaces were then immersed in 2% (v/v) GDA in 1×PBS buffer solution (pH 7.2) for 2 h under gentle stirring at 25 °C. The surfaces were rinsed 3 times with sterile 1× PBS to remove unreacted GDA. Functionalized (NH₂-GDA) surfaces were immersed in PslG_h solution (80 μg/mL in 1x PBS buffer) and incubated overnight at 4 °C then washed several times with 1× PBS buffer to remove the unbound enzyme. The concentration of immobilized enzyme was determined from the difference between the concentration of PslG_h in solution prior to immobilization minus the amount of protein that remained in the solution after the immobilization and washings steps. The enzyme concentration before and after immobilization and in washing buffers was measured using the Bradford protein assay (B6916, Sigma) with bovine serum albumin as a standard.

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).

2.5. Physical and chemical surface characterization

Surface wettability measurements and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR, PerkinElmer Spectrum 100) were used to monitor each step of the process including the surface activation (hydroxylation), silanization (APTMS), cross linking (GDA), and enzyme covalent binding (correlated with the spectra of native components). After a background scan, spectra were collected from 4000 to 625 cm⁻¹ using 64 scans at 4 cm⁻¹ resolution. The reflection intensities were corrected to compensate for their frequency (wavenumber) dependence (i.e., neglect the effect of frequency). Surface wettability of ‘wide’ tubing segments was assessed by measuring the static water contact angle using the sessile drop method. Tubing segments were cut longitudinally into two halves and a 1 μL drop of dH₂O was placed on the lumen surface and imaged. The angle between the horizontal plane and the tangent to the drop at the point of contact with the surface was analyzed by ImageJ ⁵⁴ using the contact angle plugin ⁵⁵. For thin PE-100 catheters, the lumen surface wettability was evaluated by lowering the catheters into identical vials half filled with distilled water and measuring the water level change inside the lumen. To evaluate the surface chemistry of the lumen, tubing was cut longitudinally into small, low curvature segments and the interior surface (lumen) was pressed firmly against the face of the ATR crystal to improve surface contact and obtain high quality absorbance/transmission signals.

2.6. Antibiofilm activity under static culture conditions

PAO1 cells were grown in LB medium at 37 °C overnight with shaking at 200 rpm. Cell cultures were normalized to an OD_{600 nm} of 0.5 and diluted in LBNS (1:100). 5 mL of the diluted culture was added to sterile 6-well polystyrene microplates (Thermo Scientific cat. no. 243656) containing the tested catheters segments (2.5 cm). The culture plates were incubated for 24 h at 25 °C. After incubation, catheters were removed, drained, and rinsed 3x with 10 ml of sterile 1× PBS buffer (pH 7.2) to remove non-adherent cells and media. Under strict sterile conditions, the catheters were cut into 1 cm segments and transferred to separate sterile vials for Sytox Green, crystal violet (CV) staining and colony forming units (CFU) counts, respectively.

2.7. Antibiofilm activity in vitro under dynamic flow conditions

P. aeruginosa PA01 biofilms were grown in a lab-made ‘two catheters’ device, in which the sterile untreated (control) and PslG_h bound PE-100 (10 cm) were installed together with one inlet and one outlet device (Figure S1). P. aeruginosa PAO1 was grown in LB Broth overnight, with shaking at 37 °C. The culture was then diluted 1:100 into 150 mL of fresh LBNS Broth in a 250 mL Erlenmeyer flask and stirred. The device inlet was connected to the inoculated media, and the outlet was connected to a tube mounted into a peristaltic pump (Cole Parmer) head and into a waste media bottle. The experiment began once the inoculated medium started flowing from the end of PE-100 catheters, and the flow was stopped to allow for cell attachment and biofilm growth under static conditions. After 24 h, under aseptic conditions the Erlenmeyer flask was disconnected and 20 L of diluted (1/10) and sterilized LBNS media in a container was connected to the device and media flow (0.4 mL/min) was started. LBNS media was pumped through the reactor from the top, and the waste was dispensed from the side to ensure removal of all free, unattached cells and allow the attached cells to form biofilms under flow conditions for 14 d. A filtered port was used to provide air to the bacteria. A 1 cm section of each tubing was taken daily under sterile conditions and used in a crystal violet (CV) assay, and for SYTOX Green and CFU measurements. For PE catheters, multiple 10 cm sections of the control and PslG_h-bound were connected between two multichannel devices. In this case, after long-term continuous flow (~14 d), a whole catheter was disassembled and cut into sections under sterile conditions for biofilm analysis (CV, fluorescence microcopy and CFU).

2.8. Biofilm inhibition in vitro assay

To examine biofilm inhibition using a microtiter plate assay, P. aeruginosa strain ATCC27853 was grown at 37 °C overnight with shaking at 200 rpm. The cultures were normalized to an OD₆₀₀ of 0.5 and then diluted 1:100 in LBNS. Diluted culture was added to sterile 96-well polystyrene microtiter plates (Thermo Scientific cat. no. 243656), and varying concentrations of PslG_h (0.1 to 10 μM) were added. The cultures were incubated statically for 24 h at 37 °C to allow for biofilm formation. To eliminate edge effects, ~200 μL of sterile water was placed in all outside wells, and the plate was sealed with parafilm. After incubation, nonadherent cells and media were removed by thoroughly washing the plate with deionized water. The wells were stained with 150 μL of 0.1% (w/v) crystal violet for 10 min and then rinsed with water. The remaining dye was solubilized by addition of 150 μL of 95% (v/v) ethanol and left for 10 min, after which the absorbance was measured at 595 nm using a SpectraMax M2 spectrophotometer (Molecular Devices). The amount of biofilm was proportional to the absorbance from staining with crystal violet. Experiments were performed in triplicate and all statistical analysis were performed using GraphPad Prism.

2.9. Antibiofilm activity in vivo

P. aeruginosa ATTC 27853 biofilm formation on coated catheters in vivo was evaluated using a rat venous catheter model previously developed to study Candida albicans biofilm formation 63. Pathogen-free female Sprague-Dawley rats weighing 400 g (Harlan Sprague-Dawley, Indianapolis, Ind.) were used. Animals were maintained in accordance with the American Association for Accreditation of Laboratory Care criteria, and all studies were approved by the institutional animal care committee. Briefly, 24 h after implantation, the jugular venous catheters (PE-100) were inoculated (10⁷ cells/mL) and allowed to dwell for 6 h before washing excess inoculum. Rats were sacrificed after 24 h and the catheters collected for bacterial enumeration or imaging by scanning election microscopy (SEM)⁵⁶.

2.10. Cell viability assay

To quantify viable bacteria as colony forming per units (CFU), catheters segments were immersed in 10 mL of 1× PBS, sonicated at low power for 5 min and vortexed for 15 s to release viable cells. Aliquots were diluted 10²-to10⁸-fold, and 100 μL of each dilution was plated in duplicate on LB plates. The number of colonies was counted after incubation at 37 °C for 24 h.

2.11. Fluorescence imaging of attached bacteria and biofilm

After catheter segments were rinsed with 1×PBS at pH 7.2, the adherent bacteria were fixed by 2.5% (v/v) glutaraldehyde (GDA) in 1×PBS solution for at least 1 h, then rinsed 3 times with 1×PBS to remove unreacted GDA. After fixation, samples were treated with Tween-20 (0.05% (v/v) in PBS buffer) for 10 min, rinsed with sterile PBS, and stained in the dark with SYTOX green (Life Technologies, CA, USA) for 30 min ^{37, 56, 62}. Samples were imaged by fluorescence microscopy (Olympus BX63, Tokyo, Japan) using a 20X dry objective. The green fluorescence of the biofilm was observed through a GFP filter (λ_ex/λ_em 395/470 nm). The fluorescence densities of biofilms were quantified using computerized image analysis with Olympus CellSens micro imaging software. Each data point represents the average of six images. Error bars represent standard deviation from the mean (n = 6).

2.12. Crystal violet staining of biofilms

Crystal Violet (CV) staining was used to visualize the biofilm biomass on catheters. Briefly, after the catheters were rinsed with 1× PBS buffer and air dried for 1 h at 25 °C, they were stained with 300 μL of 0.5% (w/v) CV for 30 min, then rinsed with dH₂O. CV-stained catheters segments were air dried and photographed.

2.13. Scanning electron microscopy

Catheters segments were rinsed with 1× PBS buffer and placed in fixative (1% (v/v) GDA and 4% (v/v) formaldehyde) overnight. The samples were rinsed in 1× PBS buffer two times and then placed in 1% (v/v) osmium tetroxide for 30 min, followed by immersion in hexamethyldislilazane (Polysciences, Inc., Warrington, Pa.). The samples were subsequently dehydrated in a series of ethanol washes (30% (v/v) for 10 min, 50% for 10 min, 70% for 10 min, 95% for 10 min, and 100% for 10 min). Final desiccation was accomplished by critical-point drying (Tousimis, Rockville, MD). Specimens were mounted on aluminum stubs and sputter coated with gold. Samples were observed in a scanning electron microscope (Hitachi S-5700) in the high-vacuum mode at 10 kV. The images were processed for display by using Adobe Photoshop version 5.0.

3. Results

3.1. Immobilization of PslG_h on polymer tubing by covalent binding

In our previous study, we covalently immobilized PslG_h to flat glass, PDMS and PS squares, to demonstrate that a continuous enzymatic degradation of Psl disrupted P. aeruginosa attachment and biofilm formation⁵¹. Extending this approach to medically relevant devices, we first tested conditions for uniform immobilization of PslG_h to the lumen surface of catheters of reasonable length (>10–20cm), where physical access is difficult. The materials of commercial catheters, PE, PDMS and PU, are useful for their chemical inertness (in addition to low friction coefficient, strength, and sterilizability), but also make them difficult to chemically functionalize.

For PslG_h immobilization, we used a 4-step process: surface hydroxylation by plasma oxidation; amine-functionalization by APTMS; GDA cross-linking and PslG_h covalent binding (Figure 1A)⁵¹. Surface hydroxylation is the key step in PslG_h immobilization to generate active sites (e.g. OH and COOH) for APTMS⁵⁷. The plasma treatment time to hydroxylate PE surface was tested first for simple PE sheets (1cm²), and their surface wettability was evaluated by water contact angle. Treating PE sheets with 5 min plasma exposure significantly reduced the contact angle from 92° ±2 (hydrophobic) to 21° ±1 (hydrophilic) (Figure S2A), indicating an increase in surface polar groups. ATR-FTIR confirmed a weak O-H broad band at ~3400 cm⁻¹ (Figure S2B). The low intensity of the O-H signal is due the thin surface layer and the limited sensitivity of ATR-FTIR ⁵⁷. For PDMS and PU sheets, atmospheric plasma treatment for 5 min also caused a reduction in contact angle from 108° ±4 and 69° ±2 to 21° ±3 and 20° ±1, respectively.

Tubing segments (0.5 cm, ID) of PE, PDMS and PU were then treated with plasma for 5 min, silanized with APTMS and the PslG_h cross-linked with GDA. The success of chemical immobilization along tubing length was confirmed by ATR-FTIR after each step (Figure 2A–C). ATR-FTIR spectrum of the lumen surface of untreated (control) PE tubing (Figure 2A) showed three ethylene bands at 3000–2800 cm⁻¹ (CH₂ asymmetric and symmetric stretching), 1500–1400 cm¹ (1473 and 1463 cm⁻¹ bending and wagging deformation at 1366) and 730–720 cm⁻¹ (rocking). After plasma treatment, we observed a small O-H broad band centered at ~3400 cm⁻¹ of the hydroxyl (OH) groups⁵⁶. Addition of APTMS shows FTIR spectra with peaks belonging to C-H (~2900 cm⁻¹) and N-H (~3300 cm⁻¹) stretching vibrations, and peaks corresponding to hydroxyl groups (~3400 cm⁻¹) and C=O (~1635 cm⁻¹) stretching vibrations after the addition of GDA. After incubating with PslG_h, the spectrum showed a signal at 1635 cm⁻¹ associated with the carbonyl (C=O) vibrations of an amide I band, and a broad band of O-H and N-H stretching vibrations centered at 3343 cm⁻¹. These signals were similar to those obtained from the IR analysis of pure PslG_h in buffer solution ⁵¹. For PDMS and PU tubing, similar modifications in the surface chemistry were detected by ATR-FTIR (Figure 2B and C) confirming the attachment of PslG_h to these two surfaces. Collectively, these surface characterizations confirm successful covalent immobilization of PslG_h to the lumen surface of PE, PDMS and PU tubing.

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.

3.2. Immobilization of PslG_h on PE-100 microcatheters

After PslG_h immobilization on wide tubing (0.5 cm, ID), we next tested short segments (2.5 cm) of medical grade PE-100 microcatheter (0.8 mm, ID). As shown in Figure 2D, we could measure changes in lumen hydrophobicity by the relative capillary rise of water due to capillary action ⁵⁸. The capillary height for the control PE-100 catheters was ~ 1 cm below the water level (Figure 2D) due to its hydrophobic surface. Plasma treatment increased the water level by ~ 2 cm, showing a hydrophilic character. Adding APTMS, the water level decreased below the surface level due to the increased number of hydrophobic C-H and N-H groups on the surface (Figure 2D), while GDA increased the capillary rise again due to the hydrophilic hydroxyl (O-H) and carbonyl (C=O) groups. After PslG_h immobilization, the lumen surface maintained its hydrophilic character.

The average length of a CVC catheter insertion is 11–14 cm ⁹, so for the in vitro and in vivo-testing we prepared 14 cm lengths of PE-100 medical grade tubing with PslG_h. As the lumen of a longer catheter is not directly exposed inside the plasma treatment chamber, the efficiency of hydroxylation may be affected. To test the uniformity along the tubing length, the catheters were cut into 4 equal segments after each immobilization step, and their hydrophobicity and surface chemistry measured by capillary rise and ATR-FTIR, respectively. We found that plasma treatments of < 10 min resulted in variations in the level of water reached within each segment (S1-S4) of the lumen, but exposures of 10 min or more resulted in uniform hydroxylation with even capillary rise for all 4 segments (Figure 2E). Importantly, identical water level (indicative of hydrophilicity) (Figure S3A) and ATR-FTIR spectra (indicative of surface chemistry) (Figure S3B) were also observed for all 4 sections after PslG_h immobilization, suggesting that PslG_h was also evenly immobilized along the entire length of these PE-100 catheters.

3.3. PslG_h modified polymer tubing inhibit biofilm formation

We first tested the resistance of covalently-bound PslG_h on PE, PDMS, PU tubing to P. aeruginosa PAO1 biofilm formation under static culture conditions. Fluorescence microscopy using SYTOX green was used to visualize attached bacterial cells and biofilm formation after 24 h growth. While dense biofilms were observed on the three untreated controls, those with bound PslG_h showed significantly lower cell attachment and colony formation (Figure 3A). Image analysis (cell count/cm²) revealed that the bound PslG_h significantly reduced the number of attached cells by at least 2-log (Figure 3B). These results confirm the uniformity of bound PslG_h along the lumen length of PU and PDMS and that the surfaces can effectively inhibit the adhesion, colonization and subsequent biofilm formation of P. aeruginosa PAO1.

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.

3.4. PslG_h modified PE-100 microcatheters inhibit biofilm formation

Previously, we found that the enzymatic activity of covalently-bound PslG_h on glass surfaces was dependent on the enzyme surface density⁵¹. To maximize the immobilized PslG_h surface density on the lumen surface of PE-100 microcatheters, we varied the PslG_h concentration (0, 20, 40, 80 and 160 μg/ml) in the immobilization buffer. The surface density of bound PslG_h was determined from the difference between its concentration before and after immobilization using the Bradford protein assay ⁵¹. The amount of surface-bound PslG_h increased exponentially with increasing PslG_h concentration in the buffer up to a PslG_h concentration of 80 μg/ml, at which point the maximum immobilization capacity is likely reached (Figure 4A). The maximum PslG_h surface density was estimated to be about 1.86 μg/cm². Fluorescence microscopy images showed a thick biofilm developed on the lumen surface of short (2.5 cm) untreated PE-100 microcatheter after exposure to P. aeruginosa PAO1 under static culture condition for 24 h (Figure 4A). However, a moderate reduction in the biofilm growth was observed even at a low density (1.18 μg/cm²) of surface-bound PslG_h, and complete inhibition of bacterial attachment and biofilm formation was observed at a PslG_h surface density of 1.78 μg/cm² and above (Figure 4A).

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.

To ensure that the anti-biofilm activity was due to the bound PslG_h specifically, we tested the effects of surface functionalization with APTMS and GDA on P. aeruginosa biofilm formation. The number of bacterial cells attached to APTMS (1.7 ×10⁶ CFU/cm²) and GDA (2.7 ×10⁶ CFU/cm²)-treated lumen surfaces after 24 h were not significantly different from that of the untreated microcatheter lumen (2.3 ×10⁶ CFU/cm²). However, a ~3-log reduction in attached cell numbers was observed with PslG_h-immobilized vs untreated microcatheters (1.7 ×10³ vs 2.3 ×10⁶ respectively, Figure 4B). In addition, SYTOX green (SG) and crystal violet (CV) staining demonstrated the formation of thick biofilm on the lumen surface of untreated microcatheters and those treated with APTMS or GDA, while biofilms were absent from the lumen of PslG_h-treated PE-100 microcatheters (Figure 4B). Combined, these results demonstrate that the anti-biofilm activity of the PE-100 microcatheters against P. aeruginosa is due to the immobilized PslG_h rather than other effects of the immobilization process on the microcatheter lumen surface.

To test the activity of the modified PE-100 microcatheters against clinical strains, we used P. aeruginosa strain ATCC 27853, a clinical strain with great ability to produce a more robust biofilm compared to the lab PAO1 strain⁵⁹. We first tested the ability of PslG_h in solution to inhibit its biofilm formation. Our results demonstrate that the PslG_h in solution is effective at inhibiting P. aeruginosa ATCC 27853 biofilms with an EC₅₀ of ~ 1 nM (Figure S4A). Fluorescence microscopy images of the lumen surface of untreated PE-100 microcatheter showed a thick biofilm after exposure to P. aeruginosa ATCC 27853 under static culture condition for 24 h (Figure S4B). However, a significant reduction in the number of attached cells (~3-log) was observed compared to the control (Figure S4C).To ensure the activity of immobilized PslG_h along the entire microcatheter length (10 cm), we tested the anti-biofilm activity against P. aeruginosa PAO1 biofilm under 24 h static culture. After incubation, CV staining of biofilm mass along the 10 cm microcatheter showed almost no biofilm formation on the PslG_h-bound lumen surface, whereas a heavily CV-stained biofilm appeared within the untreated PE-100 control catheter after 24 h (Figure 5A). To test the uniformity of the anti-biofilm activity along the entire 10 cm catheter lumen, catheters were cut into ten 1-cm long segments after incubation in the same conditions as above. Then, 5 alternating segments (Figure 5B) were tested for resistance to P. aeruginosa PAO1 biofilm formation using fluorescence microscopy (S2, S4, S6, S8, S10) and CFU (S1, S3, S5, S7, S9). Microscopic images showed thick biofilms within all 5 segments of the untreated catheter lumen (Figure 5C). In comparison, the covalently-bound PslG_h significantly eliminated biofilm formation of P. aeruginosa for all PE-100 segments (Figure 5C). Quantitative culture confirmed that the PslG_h-treatment of catheters significantly reduced attached cell density from 6.5–6.8 ×10⁶ CFU/cm² (untreated control) to 1–8.3 ×10³ CFU/cm² (PslG_h-bound) after 24 h, representing a ~3-log reduction in CFU (Figure 5D). These results confirm we succeeded in generating a uniform, high density PslG_h coating of the lumen surface, with strong anti-biofilm activity against P. aeruginosa along the length of these medical grade PE-100 catheters.

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.

3.4. In vitro flow culture

We next tested the PslG_h-modified catheters in an in vitro flow culture model (Figure S1). P. aeruginosa cells were allowed to adhere under static culture conditions for 1 day after which a constant flow (0.6 mL/min) of fresh LBNS media was passed through the lumen of both untreated control and PslG_h-bound PE-100 catheters in parallel. Fluorescence microscopy (Figure 6A) demonstrated that the PslG_h-bound catheters were resistant to biofilm formation on the lumen surface for the 14 d test period under dynamic culture flow conditions, as compared to the control PE-100 catheters. The rate of cell attachment and biofilm formation was also measured under these flow culture conditions. Quantitative culture confirmed that PslG_h-bound catheters exhibited an ~ 3 log reduction in attached cell numbers during the first 11 d of flow, as compared to untreated catheters. Bacterial counts for the untreated catheters decreased between days 11 and 14, which resulted in a reduction of only 2 log between the treated and control catheters at day 14 (Figure 6B). Taken together, these results indicated that PslG_h immobilized on the catheter lumen surface inhibits biofilm growth for at least 14 d under dynamic flow culture conditions.

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.

3.5. Antibiofilm activity of PslG_h modified catheter in presence of serum

As a prelude to an in vivo study, we investigated the effect of blood proteins, storage stability and catheter sterility on the antibiofilm activity of the PslG_h-bound catheters. Immediately after catheterization, the surface of indwelling catheters becomes covered with plasma proteins that could potentially interfere with the function of the immobilized enzyme. Therefore, the activity of the lumen-bound PslG_h was tested after being treated with serum for 24 h. In general, there were significant increases in the number of cells that adhered on both untreated and treated catheters after serum treatment (Figure S4). Nevertheless, CV staining (Figure S4A) and SYTOX green (Figure S4B) both confirmed a reduction in bacterial biofilm biomass on the lumen surface of PslG_h-bound catheters relative to the untreated control catheter, despite treatment with serum. A few cell clusters were observed by fluorescence microscopy of the PslG_h-bound catheter lumen (Figure S4B). Quantitative culture showed that, relative to the control catheters, immobilized PslG_h reduced attached cell density within the catheter lumen before and after serum treatment by 3-log and ~2.5-log, respectively, (Figure S4C). Our results show that while serum proteins increase the adherence of cells to both the control and enzyme-treated surfaces, the PslG_h-bound PE catheters remain more resistant to cell attachment even in the presence of serum.

3.6. Sterility and shelf-life storage of the catheter and PslG_h modified catheter

Immobilization procedure (Figure 1) was carried out under aseptic conditions. Prior to immobilzations, caatheters were either pre-sterilized using70% ethanol or ethylene oxide. The immobilzation steps involve the use of atmospheric plasma, 80% ethanol, 2% GDA and filter sterilized PslG_h in PBS buffer which are sufficient to maintain the sterility of catheters during immobilization. To confirm, we tested the sterility of PslG_h-bound catheters with a standard plate counting method. Results confirmed the absence of contamination.

In addition, we tested the storage stability of PslG_h-bound catheters after storage for 30 d at 4 °C under both dry and wet conditions. PslG_h-bound catheters were found to remain resistant to biofilm formation as shown from fluorescence microscopy (Figure S6A) and their corresponding cell counts, which exhibited a 2.5–3 log reduction (Figure S6B).

3.7. PslG_h modified catheter inhibits biofilm formation in vivo

Finally, we tested the anti-biofilm activity of these PslG_h-bound PE-100 catheters in the complex environment found in vivo, using a central venous catheter infection (biofilm) rat model ⁶⁵. In this model, the clinical strain P. aeruginosa ATCC 27853 was chosen as it was isolated from a hospital blood specimen and has been shown to produce a more robust biofilm compared to the lab PAO1 strain ⁵⁹. Twenty-four hours after intravascular placement, SEM imaging of untreated catheter segments (Figure 7A) showed a dense biofilm network that consists of a relatively large number of cells enclosed in an extracellular matrix. In comparison, segments from the PslG_h-bound catheter contained scattered and weakly attached cells and a lack of biofilm matrix. Quantitative culture of the PslG_h-bound catheter showed a significant inhibition of biofilm formation 24 h after infection, as compared to the untreated catheters (~2 log CFU reduction) (Figure 7B).

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.

4. Discussion

Developing robust approaches to limit bacterial biofilm formation on biomaterials through non-fouling (non-biocidal) mechanisms remains relatively new for antimicrobial biomedical devices. Antimicrobial strategies that do rely on biocidal drug release ⁶⁰ are generally ineffective over time as they cannot prevent surface colonization (new cells adhering to attached dead cells), as evidenced by the high rates of biofilm-related disease that persist globally ^{4, 39}. Since Psl adsorption plays a key role in the mechanism of P. aeruginosa cell attachment and biofilm formation, our approach specifically targets and disrupts this microbial adhesion mechanism. This approach differs from non-specific mechanisms to non-fouling such as SLIPS (slippery liquid infused porous surfaces) ^61–62, and other non-wetting surfaces. For well-known, common pathogens it seems advantageous to target cell attachment, at the early stages of biofilm development. It has also been reported that, in mixed species biofilms, Psl can also contribute to the protection of other pathogens such as Escherichia coli and Staphylococcus aureus from antimicrobials¹⁵. Therefore, specific, selective prevention of P. aeruginosa biofilm formation should not only help enable antibiotic therapies to eradicate infections and reduce the development of acquired resistance, but also help the host immune system to eliminate infections associated with medical devices that are caused by both biofilm and non-biofilm producers.

The immobilization of the PslG_h glycoside hydrolase uniformly along the full length of these medical grade, small-diameter PE catheters, has been a significant technical achievement due to the inert nature of PE, and the physically confinement of access to the lumen surface (inside a 1mm tube, 14 cm long) for the plasma and solution penetration. The lumen surface of implanted catheters can become exposed to blood and body fluids, and has a high risk of biofilm infection. Previous efforts to functionalize catheter lumen surfaces, such as uniform activation with polar groups, was shown to be a challenge ⁶³. The narrow PE-100 catheters used herein thus represent a kind of ‘worst case’ application for medical devices, and our results suggest that a wide range of device sizes, material types and morphologies should be possible for enzyme immobilization.

A key finding in our work here is stability. The results show a consistent, stable activity of these enzymes to disrupt biofilm formation (through continuous Psl polysaccharide degradation) under in vitro flow conditions for extended periods (14 d), after extensive sterilization and storage (30 d, 4°C), and in the complexity of an in vivo environment. These results suggest that PslG_h remains active after surface immobilization, which may make them suitable for a wide range of medical devices. Importantly, the immobilization procedure helped to maintain sterility of the catheters as conventional methods such as ethylene oxide did not work as the sterilized PslG_h surfaces lost their anti-biofilm activity (Figure S5). The difference between the CFU log reduction observed in vivo (Figure 7B) and that observed in the in vitro assay (Figure 6B) likely reflects the enhanced biofilm formation capacity of strain ATCC 27853 ⁵⁹ as well as the effect body fluids and blood serum on promoting the biofilm growth ⁶⁴. Overall, our work demonstrates that covalent immobilization of enzymes on biomedical tubing surfaces can represent an effective and feasible design strategy for non-fouling antimicrobial material design. These represent a ‘bio-active’ interface to specifically prevent bacterial adhesion mechanisms through continuous hydrolytic activity of a biofilm matrix component.

5. Conclusions

In summary, we have generated a sterile, uniform, and high-density surface of immobilized PslG_h enzyme throughout the lumen of long, small diameter, medical grade catheters, in an effort to improve the performance of indwelling catheters against microbial biofilm growth. The catheters were highly effective at preventing P. aeruginosa biofilm growth under both static and dynamic flow conditions in vitro, and even for long periods of time (11 days). Finally, preliminary in vivo results show the PslG_h-bound catheters dramatic resistance to biofilm growth in the complex environment of an infection model. These results demonstrate for the first time that ‘bio-active’ enzyme-modification of surfaces can realistically be applied to a wide range of biomaterials, uniformly over large areas, and with long-term activity. There is a wide range of biomedical devices that may benefit from these effects on P. aeruginosa biofilm growth, to significantly impact the problem of hospital acquired infections, complications and costs ⁵⁴.