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. 2025 Dec 5;26(3):e00501. doi: 10.1002/mabi.202500501

Polydopamine‐Antibiotic Composite Coating for Antibiofilm Applications

Gillian A Kropp 1, Hannah Q Karp 3, Jayasimha Rao 3,4,5,6, Henriette Suzanne Muller 1, Nammalwar Sriranganathan 6,7, Elizabeth S Nowak 3,4,5,6, Michael D Schulz 1,2,6,✉
PMCID: PMC12993261  PMID: 41350243

ABSTRACT

Urinary catheterization is a common procedure, affecting 15%–25% of hospitalized patients. This procedure, however, often results in bacterial infections, primarily caused by biofilm‐forming Gram‐negative bacteria that adhere to the catheter surface. To address this challenge, we developed a polymer‐based antimicrobial coating using polydopamine (PD) embedded with gentamicin (Gent). We evaluated the antibiofilm efficacy of this coating (PD‐Gent) using a biofilm‐forming isolate of Pseudomonas aeruginosa (PAO1) as P. aeruginosa is commonly implicated in catheter‐related infections. We observed that the PD‐Gent coating significantly reduced biofilm formed by PAO1 compared to the uncoated control. Importantly, the coating maintained its antibiofilm activity across diverse substrates, including polystyrene, poly(vinyl chloride), and silicone. The approach was further extended to incorporate other antibiotics (tobramycin, amikacin), demonstrating adaptability to multiple antimicrobial agents. Finally, artificial urine inoculated with PAO1 was deployed through PD‐Gent–coated silicone Foley catheters under continuous flow for 24 h. Despite this continuous introduction of PAO1, the coated catheters inhibited biofilm formation by three‐fold compared to the uncoated control catheters. These findings underscore the promise of PD‐Gent as a robust, versatile coating with strong potential to significantly reduce the incidence of catheter‐associated infections in clinical settings.

Keywords: antibiofilm catheter, biofilm, catheter coating, CAUTI, gentamicin, polydopamine

Antibiotic‐loaded polydopamine coatings are developed to combat biofilm‐forming bacteria on catheter surfaces. These coatings strongly inhibit Pseudomonas aeruginosa biofilm formation on diverse materials, including during continuous artificial‐urine flow, and can accommodate multiple antibiotics. Together, these features establish a promising pathway to reducing catheter‐associated infections in clinical settings.

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1. Introduction

Catheter‐associated urinary tract infections (CAUTIs) are the most common hospital‐acquired infection, accounting for more than 75% of all urinary tract infections and representing a major biofilm‐related clinical challenge [1, 2]. CAUTIs are often treated with systemic antibiotics and catheter replacement; however, systemic antibiotic use is not recommended for prophylactic purposes [3, 4]. As a result, new strategies for addressing CAUTIs and other biofilm‐related infections are urgently needed. Several strategies are currently employed to prevent or mitigate biofilm‐driven device‐associated infections, including early and frequent device removal, systemic antibiotics, and antimicrobial lock therapy (i.e., local instillation of antibiotics into the lumen of a venous catheter) [5]. However, these approaches are often ineffective and, in some cases, contribute to the emergence of antimicrobial resistance [6, 7, 8, 9]. More recently, antimicrobial catheter coatings have been investigated as a complementary strategy. Polymer‐based coatings have proven particularly promising, leveraging decades of research on polymers for medical applications, including drug‐delivery vehicles, drug‐capture devices, and tissue engineering scaffolds [10, 11, 12, 13, 14, 15].

Among polymeric materials, polydopamine (PD) has attracted attention due to its strong surface adhesion and versatility in forming thin conformal films or nanoparticles (PDNPs) capable of encapsulating small molecules for drug delivery [16, 17, 18, 19, 20]. Despite these advantages, PDNP‐based systems face challenges in scalability and dispersity control, limiting their clinical translation. Antibiotic–silver nanoparticle (AgNP) composites have also been explored, but AgNPs are prone to aggregation and exhibit dose‐dependent cytotoxicity [21, 22, 23, 24]. Importantly, many pathogens associated with CAUTIs require combination therapies that leverage multiple antibacterial mechanisms [6, 9]. However, few coatings have demonstrated the ability to incorporate multiple drugs or to be broadly applied across different substrates.

To address these limitations, and inspired by established uses of PD in the biomedical field [25, 26], we developed a catheter coating that harnesses PD's adhesive properties to immobilize antimicrobial agents directly onto the catheter surface. The goal of this work was to establish a facile coating strategy compatible with commercial catheters, capable of incorporating multiple antibiotics, and stable under urinary flow conditions. This PD–antibiotic composite coating eliminates the challenges associated with nanoparticle synthesis while maintaining effective drug loading and surface adhesion. Specifically, we synthesized a PD coating containing the aminoglycoside gentamicin (Gent) and further demonstrated that the approach is extendable to other antibiotics (tobramycin and amikacin). Moreover, the PD–Gent coating remains effective across multiple medically relevant surfaces, underscoring its potential as a broadly applicable strategy for reducing device‐associated infections.

2. Results and Discussion

PD coatings were formed through the oxidative polymerization of dopamine hydrochloride under mildly basic conditions using Tris buffer (Figure 1A) [27]. This process, which occurs under biocompatible and reagent‐free conditions, is well‐suited for modifying medical device surfaces without compromising material integrity or safety [26]. The mechanism is thought to involve an initial step where pH‐induced auto‐oxidation of dopamine forms quinone intermediates, followed by crosslinking (through biphenyl bond formation to produce polycatecholamines) and subsequent cyclization [26]. The resulting coating exhibited strong adhesion to a variety of substrates due to the presence of reactive catechol and amine groups [27]. These same functional groups also enable antibiotic incorporation through electrostatic, hydrogen bonding, and π–π interactions [23]. In this study, gentamicin was introduced during coating formation, enabling its incorporation into the PD matrix and generating a stable antimicrobial surface. To simulate catheter surfaces, coatings were applied to poly(vinyl chloride) (PVC) tubing, a material commonly used in catheters and other medical devices [4, 28].

FIGURE 1.

FIGURE 1

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(A) Reaction scheme for dopamine polymerization on PVC tubes (B) Optical microscopy images (11.2x magnification) of uncoated PVC control tubes and PD‐Gent–coated tubes made using 1.0 mg/mL dopamine solution during polymerization (scale bar = 1 mm).

Scanning electron microscopy coupled with energy‐dispersive X‐ray spectroscopy (SEM‐EDX) was used to evaluate the surface characteristics of the coating on the PVC tubes. However, due to the sampling depth of SEM‐EDX exceeding the expected coating thickness, neither the coating depth nor heterogeneities could be resolved. Optical microscopy revealed a visible color change on the PVC surface (Figure 1B), indicating that the PD coating remained localized on the surface and did not penetrate the entire thickness of the PVC tube. Additionally, PD‐Gent–coated tubes exhibited a distinct dark layer compared to uncoated PVC controls (Figure S1). Characterizing PD‐based coatings on flexible, porous materials like PVC and catheter tubes remains challenging because PD aggregates tend to deposit within surface pores, complicating surface‐specific analyses [29]. However, the physical appearance of the PD‐Gent coating was consistent with other PD‐based materials in the literature [16, 30, 31].

2.1. PD‐Gent Coating Development

To optimize gentamicin incorporation into the PD‐based coating, we investigated how a range of dopamine concentrations in the polymerization solution affected bacterial viability when exposed to the resulting coating. Dopamine hydrochloride (1–10 mg/mL) was polymerized in the presence of gentamicin (2 mg/mL) and PVC tubes in Tris buffer. Adjusting the initial dopamine concentration enabled control over the gentamicin content in the resulting composite coating (Figure 2). Gentamicin remaining in solution after coating formation was quantified using liquid chromatography–mass spectrometry (LC‐MS), and the difference was used to calculate the amount that was incorporated into the coating.

FIGURE 2.

FIGURE 2

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Gentamicin incorporated into the composite coating on a 25 cm PVC tube determined by LC‐MS. Data points represent the mean, error bars represent standard deviation (n = 4).

Gentamicin attachment steadily increased as the dopamine monomer concentration increased from 1 to 4 mg/mL; however, attachment systematically decreased when the polymerization was performed using 5–10 mg/mL of dopamine (Figure 2). Once gentamicin attachment was confirmed, the materials were further assessed for antibacterial activity using a laboratory strain of P. aeruginosa (PAO1) and a gentamicin‐resistant strain, P. aeruginosa 383‐rahU::GM [32]. We initially hypothesized that coatings with the most gentamicin attached (synthesized when dopamine concentration was 4 mg/mL) would result in the highest antimicrobial activity due to a higher amount of the antimicrobial agent in the coating. Despite the high gentamicin content, bacterial growth inhibition was unsuccessful at high dopamine coating solution concentrations (Figure 3). In fact, when PAO1 was exposed to the coating for 24 h and evaluated for bacterial viability, a bactericidal effect was observed only with coatings made using 1.0 mg/mL dopamine hydrochloride solution (Figure 3). The reduced efficacy against PAO1 is likely due to the formation of more compact PD aggregates at higher dopamine concentrations [33], which may hinder gentamicin from interacting with the bacteria. As expected, no bacteriocidal effect was observed when PAO1 was incubated with PVC tubes coated without gentamicin (PD‐only). Similarly, we observed no bactericidal effect of the PD‐Gent coating against the gentamicin‐resistant strain 383‐rahU::GM (Figure 3).

FIGURE 3.

FIGURE 3

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Viability of PAO1 and 383‐rahU::GM after incubation with PVC tubes coated with either PD‐Gent or PD‐only at varying dopamine concentrations (1–10 mg/mL).

Based on these PAO1 growth data, we synthesized coatings using dopamine concentrations less than 1.0 mg/mL (0.25, 0.50, and 0.75 mg/mL), while maintaining a gentamicin concentration of 2 mg/mL in the polymerization solution. We observed negligible differences in gentamicin incorporation into the composite at these low dopamine concentrations (Table S1). We subsequently assessed bacterial viability (see below) and noted a clear bactericidal effect of the PD‐Gent coating at each of these lower concentrations (Figure S2).

We next quantified biofilm formation on these materials. To do so, PVC tubes were rinsed with de‐ionized (DI) water before adding 60 µL aliquots of biofilm‐staining crystal violet (CV). After 10 min, excess CV was rinsed out repeatedly using sterile water, and CV‐stained tubes were air‐dried overnight. PD‐Gent–coated PVC tubes showed minimal CV staining following PAO1 incubation, indicating effective inhibition of biofilm formation. In contrast, PD‐only coated (made without gentamicin) and uncoated (control) tubes retained dark purple CV staining consistent with substantial biofilm growth (Figure 4A). We observed a substantial reduction in biofilm formation in PD‐Gent samples synthesized with dopamine concentrations ranging from 0.25 to 1.0 mg/mL, compared to PD‐only and uncoated control tubes (Figure 4B). The gentamicin‐resistant strain, 383‐rahU::GM, incubated in PD‐Gent coated tubes retained its biofilm formation capabilities, as expected.

FIGURE 4.

FIGURE 4

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(A) CV staining of PD‐only and PD‐Gent tubes using PD concentrations of 0.25 and 0.50 mg/mL vs PAO1 and 383‐rahU::GM. (B) Biofilm formation on coated PVC tubes after PAO1 incubation for 24 h. The coatings were synthesized using 2 mg/mL gentamicin and increasing PD concentrations. Data points are representative of average optical density, and error bars are standard deviation (n = 12).

To further evaluate bactericidal activity, the viability of PAO1 and 383‐rahU::GM was assessed after 24 h exposure to the PD‐Gent coating (Figure S2). The planktonic cell viability of each tube was assessed qualitatively using a 48‐pin micro‐plate pin replicator after expelling the contents of each tube into a 96‐well plate. The liquid contents of the PD‐Gent tubes showed no growth of PAO1 across all concentrations of dopamine studied. As anticipated, we observed no bactericidal effect in the gentamicin‐resistant strain (Figure S2). A control experiment was also performed in which PVC tubes were exposed to a gentamicin–tris solution (Gent‐only) for 24 h at 60°C, the same conditions as the coating protocol. These tubes were subsequently incubated with PAO1 and stained with CV, which showed similar optical density measurements to the uncoated control (Figure S3). These results indicate that PD is required for gentamicin to attach to the surface of the PVC tube.

2.2. PD‐Gent Coating Retains Antibiofilm Properties on Polystyrene

To determine if the coating material could adhere to other surfaces, we coated and assessed PAO1 biofilm formation and bacterial viability on polystyrene (PS) 96‐well plates. PD‐Gent–coated 96‐well plates were incubated with PAO1 for 24 h and were then assessed for biofilm formation and bacterial viability. We again varied the dopamine concentration in the coating solution to determine whether the previously observed trends persisted with a different underlying substrate. The minimum PD content required for sufficient gentamicin attachment and antimicrobial activity was determined by varying the dopamine concentration from 0–1 mg/mL during coating synthesis. In the Gent‐only coating (no PD), biofilm formation was comparable to that observed in uncoated (control) wells (Figure S4), again indicating that gentamicin does not effectively bind without the presence of adequate PD. At dopamine concentrations of 0.05 and 0.10 mg/mL, the biofilm formation remained unchanged compared to the uncoated well, suggesting insufficient polymer for effective gentamicin attachment (Figure S4). However, at a dopamine concentration of 0.25–1 mg/mL, a reduction in biofilm formation was observed (Figure S4). As expected, no bactericidal effect was observed using the PD‐only coating (Figure 5A). PD‐Gent coatings with dopamine concentrations between 0.25–1 mg/mL also inhibited PAO1 growth (Figure 5B). These results are congruent with the reduction in PAO1 biofilm and bacterial viability observed in PD‐Gent‐coated PVC tubes.

FIGURE 5.

FIGURE 5

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(A) Bacterial viability measured in log colony‐forming units (CFU) of PAO1 exposed to PD‐only and PD‐Gent coated 96‐well plates by dopamine concentration. Data points are the mean; error bars are representative of standard deviation (n = 12). (B) Composite image of cell‐viability assays for PD‐Gent coatings using PD concentrations of 0.05–1.0 mg/mL.

2.3. Coating Methodology Can Accommodate Other Antibiotics

Aminoglycosides are an effective treatment option for Pseudomonas aeruginosa urinary tract infections, based on their high urinary penetration and reduced risk of nephrotoxicity in this setting [34, 35, 36, 37]. Like gentamicin, tobramycin and amikacin are aminoglycoside antibiotics that are effective against gram‐negative bacteria, which are the most common pathogens in CAUTIs [38]. To evaluate the versatility of this coating methodology, PS‐well plates were coated in a PD‐Tobramycin or PD‐Amikacin composite using the same procedure as the PD‐Gent coating method, but substituting tobramycin or amikacin for gentamicin, respectively. As with the PD‐Gent–coated well plates, under static conditions, we observed successful biofilm inhibition compared to uncoated control wells (Figure 6). These results demonstrate that the PD‐antibiotic protocol is applicable to multiple antibiotics.

FIGURE 6.

FIGURE 6

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Biofilm quantification via crystal violet optical density on untreated (gray), PD‐Tobramycin (green), and PD‐Amikacin (orange) coatings using a dopamine coating solution concentration of 0.25 mg/mL. Bars represent the mean, and error bars are the standard deviation (n = 18).

2.4. Efficacy in a Dynamic Artificial Urinary Tract Model

The previous experiments involving bacterial incubation in PVC tubes assessed biofilm growth under static conditions. To more accurately simulate clinical conditions, we developed an experimental setup to test the PD‐Gent coatings on commercial urinary catheters subjected to a constant flow of artificial urine containing bacteria (Figure 7). In this system, Bardex Foley silicone catheters were connected to a reservoir (feeding bag) simulating a bladder. The bacteriuria flow was controlled by a roller clamp on the feeding bag, which delivered the bacteriuria at a constant rate (approximately 0.1 mL/min). To maintain continuous flow and prevent uncontrolled PAO1 growth, the bag was elevated outside the incubator at room temperature (Figure S5). Meanwhile, the flow inside the catheter occurred within the incubator, creating a warm environment conducive to biofilm formation, simulating physiological conditions. A similar flow experiment was conducted using DI water to determine the rate of gentamicin release. However, LC‐MS detected no gentamicin release over time, suggesting the material is either contact‐active or releasing low levels of gentamicin below the limit of detection for LC‐MS (Figure S6).

FIGURE 7.

FIGURE 7

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In vitro bladder system with silicone urinary catheters (uncoated control (light gray) and PD‐Gent‐coated (dark gray)). Bacteriuria flow through the catheter occurred in an incubator at 30°C while the bladder was hung outside the incubator.

After 24 h under flow, the PD‐Gent coating appeared stable and unaffected by the dynamic flow conditions. As anticipated, greater biofilm formation occurred in the PD‐Gent–coated catheters under flow conditions compared to static conditions, likely due to the continuous introduction of bacteria. Nevertheless, the PD‐Gent–coated Foley catheters significantly reduced biofilm formation relative to the uncoated control catheters. We observed a three‐fold reduction in optical density for the PD‐Gent coated catheters compared to the uncoated controls, representing a significant decrease in biofilm biomass from using the composite coating (Figure 8).

FIGURE 8.

FIGURE 8

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Optical density of uncoated and PD‐Gent–coated Foley catheters after undergoing constant flow of PAO1‐artifical urine solution. Bars represent means, and error bars are representative of standard deviation (n = 3), ****: p‐value < 0.0001 (Table S2).

To assess planktonic bacterial viability in coated and uncoated catheters, 30 µL aliquots of the bacteriuria were collected from the catheter outlet over time and plated on agar, followed by overnight incubation at 37°C. A significant reduction in surviving PAO1 colonies was observed after 2 and 5 h of continuous flow with exposure to PD‐Gent (Figure S8). Across the three independent trials, the inhibition of biofilm formation remained consistent while the degree of reduction in PAO1 planktonic viability varied (Figure S8). This variability is also consistent with other studies that show that gentamicin is less effective against planktonic bacteria under flow conditions [39, 40, 41, 42, 43, 44]. Nonetheless, we believe this reduction in biofilm is sufficient to indicate successful antibacterial properties. We propose that biofilm suppression results from a synergistic combination of the catheter's antibacterial surface and the continuous flow of fluid, which physically flushes bacteria from the system.

3. Conclusion

This work establishes a versatile and scalable coating strategy to combat biofilm formation on catheters. By exploiting the strong adhesive properties of polydopamine to immobilize aminoglycoside antibiotics (gentamicin, tobramycin, amikacin), we developed coatings that significantly suppressed Pseudomonas aeruginosa biofilm formation under both static and dynamic conditions. Importantly, this platform is substrate‐independent—effective on PVC, polystyrene, and silicone—and readily accommodates multiple antibiotics, highlighting its adaptability. Together, these findings position PD‐based antimicrobial coatings as a clinically relevant approach with strong potential to reduce biofilm‐associated infections across a broad spectrum of indwelling medical devices.

4. Materials and Methods

4.1. Materials

Sodium chloride, tobramycin, amikacin, calcium chloride, gentamicin sulfate, dopamine hydrochloride, urea, and bovine serum albumin (BSA) were purchased from Sigma–Aldrich. Disodium sulfate, tris base, and ammonium chloride were purchased from Oakwood. Hydrochloric acid, trisodium citrate dihydrate, sodium oxalate, and crystal violet were purchased from Thermo Fischer Scientific. Potassium phosphate monobasic was purchased from Acros. Tryptone soy broth (TSB) was purchased from Oxoid. All chemicals were used as‐is.

4.2. Characterization

4.2.1. Liquid Chromatography Mass Spectrometry (LC‐MS)

An Agilent (Santa Clara, CA) 1260 HPLC system equipped with a binary pump (1312B), autosampler (1329B), oven heater (1316B), and single quad MS as a detector was used for all separations and detections. Gentamicin was separated using an Agilent C8 column (250 mm × 4.6 mm, 5 µm particle size) under isocratic conditions with a mobile phase of 98:2 (v/v) 0.1% formic acid in water:0.1% formic acid in methanol. The flow rate was maintained at 0.8 mL/min. Most of the detections were obtained in full scan mode (50–600 amu); however, for low concentration samples, detections were obtained in selected ion monitoring (SIM) mode using ion 478.4 amu.

To determine gentamicin release, mass spectrometry analysis was performed on a Shimadzu 8060 triple quadrupole mass spectrometer interfaced with a Nextera LC‐20 UPLC (Shimadzu Corporation, Tokyo, Japan). Separation was obtained using an HSS T3 column (Waters, Milford, MA, USA), a flow rate of 0.3 mL/min, and mobile phases of acidified water (A) and acidified acetonitrile (B), each with 0.1% formic acid. A 10‐min binary gradient was employed as follows: 1% B from 0–3 min, a linear ramp to 90% B at 6 min, and return to initial conditions at 7.5 min. Injection volume was 1 µL. Multiple Reaction Monitoring (MRM) transitions were developed with positive ionization for gentamicin constituents C1, C1a, and C2, with two transitions per compound.

Parent Fragment Ion

Gentamicin C1a 450.2 322.2 Quantifier

450.2 163.3 Qualifier

Gentamicin C1 478.2 322.2 Quantifier

478.2 157.0 Qualifier

Gentamicin C2 464.2 322.2 Quantifier

464.2 159.9 Qualifier

4.2.2. Optical Microscopy

A ZEISS Axio Zoom.V16 was used to image the PD‐Gent coating on the surface of PVC tubes. The magnification used was 11.2x. Samples were sliced perpendicular to the length of the PVC tubes to show the cross‐sectional area of the coating.

4.3. Synthetic Methods

4.3.1. Poly(vinyl chloride) (PVC) Tube Sterilization

Tygon PVC tubing was cut into 25 cm segments and placed in a 500 mL round‐bottom flask to sterilize before undergoing coating. To begin sterilization, 200 mL of 200 proof ethanol and 200 mL of DI water were added to the flask, which was then sonicated for 30 min. After removing the solvent, the tubes were rinsed with DI water and dried in a 60°C oven in preparation for polymerization.

4.3.2. Synthesis of PD‐Gent Coating

PVC Tubes and Polystyrene Well‐plates: Tris‐buffer was prepared by dissolving Tris‐base (0.5 m) in DI water and adjusting the pH to 8.5. Dopamine hydrochloride was dissolved in 24 mL Tris‐base before adding gentamicin sulfate (2 mg/mL). This solution was added to a 6‐dram vial equipped with a stir‐bar and 25 cm of sterilized PVC tubing cut into 5 cm segments. The vial was heated to 60°C with magnetic stirring and removed after 22 h. PVC tubing was subsequently rinsed with DI water until the solution ran clear, followed by oven drying at 60°C. An aliquot (3 mL) of reaction solution was collected and passed through a 0.45 µm syringe filter for liquid chromatography analysis. A similar procedure was used for coating well‐plates using 150 µL solution in each well, followed by heating in an oven at 60°C.

Silicone Foley Catheters: Dopamine hydrochloride (100 mg) was dissolved in 100 mL of Tris base solution (described above), followed by the addition of 200 mg gentamicin sulfate in a 100 mL round‐bottom flask. Once dissolved, Foley All‐Silicone catheters were placed in the solution equipped with a magnetic stir bar. This polymerization flask was placed into a 60°C oil bath and allowed to stir for 22 h. After polymerization, the catheter was rinsed with DI water to remove unreacted dopamine hydrochloride and unattached PD. The coated catheter was placed in a 60°C oven to dry overnight.

4.4. Antibacterial Activity

4.4.1. Bacterial Cell Culture

A laboratory strain of biofilm‐forming P. aeruginosa (PAO1) and 383‐rahU::GM were used [32]. Single bacterial colonies were sub‐cultured in 4 mL of Luria Bertani (LB) medium overnight at 37°C and shaken at 200 rpm. Bacterial subcultures were diluted to have an OD600 of 0.01 in minimal M9+ media (supplemented with sterile 20% glucose, 1 m MgSO4, 0.1 mm CaCl2, 3% casamino acid in 8‐hydroxyquinolone solution) to establish a standard solution. Aliquots of this standard solution were further diluted to contain an inoculum of 1.7 × 107 colony forming units (CFU)/mL before pipetting into PVC tubes or PS 96‐well plates.

4.4.2. Crystal Violet (CV) Staining

Biofilm biomass was determined using an established method from the literature, which quantifies biofilm density by measuring absorbance of biofilm stained with 0.1% CV solution [32, 45, 46, 47, 48, 49, 50, 51]. The liquid content of the wells and PVC tubes was removed and used to assess bacterial viability (described below). Well plates and PVC tubes were stained with 100 and 60 µL, respectively, of a 0.1% CV solution, followed by 10 mL of sterile water to remove excess CV. The CV was solubilized in 100 µL of 33% glacial acetic acid, and absorbance was measured at 590 nm. Non‐specific binding between the PD, gentamicin, and CV solution was observed. To address this background interference, we incubated tubes and well‐plates with M9+ solution without bacteria as a control, which was used to correct for the background non‐specific binding.

4.4.3. Bacterial Viability Studies

After 24 h, the bacterial medium was diluted to assess bacterial viability. Aliquots were either pipetted (3 µL) or directly stamped onto Tryptic Soy Agar (TSA) plates and incubated at 37°C for 24 h. Colony‐forming units (CFUs) of planktonic bacteria were quantified following a four‐fold serial dilution, and CFU counts were verified by 2–3 independent observers.

4.4.4. Bacteriuria for In Vitro Artificial Urinary Tract Model

Artificial urine, containing a mixture of protein and various salts (BSA, urea, TSB, sodium chloride, calcium chloride, disodium sulfate, ammonium chloride, trisodium citrate dihydrate, sodium oxalate, and potassium phosphate monobasic), was made using previously reported methods [52]. Using the bacterial culture method described above, 40 mL of the above PAO1 dilution (1.7 × 107 CFU/mL) was added to 360 mL of artificial urine, mixed, then subsequently poured into a sterile feeding bag that simulates a bladder. During the in vitro study, 30 µL aliquots of bacteriuria were collected after passing through the catheters and were added to TSA plates. TSA plates were then placed into a 37°C incubator. These aliquots were taken in the first 5 h to evaluate early PAO1 planktonic viability in the flow model.

4.4.5. Method for Comparative Analysis of Biofilm Formation for Silicone Catheters

After 24 h of continuous bacteriuria flow, both PD‐Gent coated and uncoated control catheters were stained with CV to assess biofilm formation. Minimal biofilm accumulation was observed on the PD‐Gent–coated catheter. To assess biofilm biomass, CV‐stained biofilm was solubilized from the catheter surfaces using 5 mL of 33% glacial acetic acid, generating a solution referred to as 100% Biofilm‐CV solution (Figure S7). However, the undiluted biomass solutions from both coated and uncoated samples exceeded the spectrophotometer's detection range (OD5₉₀), preventing accurate measurement. To bring the absorbance values within the linear detection range of the instrument, a serial dilution was performed. The 100% solution was diluted stepwise (e.g., 1:2, 1:4, 1:8) until OD5₉₀ readings fell within measurable limits, enabling a comparative analysis of biofilm formation between samples (Figure S7).

Funding

This work was supported by Carilion Clinic through the Research Acceleration Program Grant #65160.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: mabi70111‐sup‐0001‐SuppMat.docx.

MABI-26-e00501-s001.docx (16.8MB, docx)

Acknowledgements

The authors gratefully acknowledge the Virginia Tech Materials Characterization Facility and Brenna Knight in the Dove Lab for access to, and support with, microscopy and surface analysis tools. The authors thank the Research Acceleration Program at Carilion Clinic for funding this study. We also thank the Infectious Diseases Department at Carilion for providing clinical insight and resources. Liquid chromatography–mass spectrometry (LC‐MS) analysis was performed with assistance from Dr. Mehdi Ashraf‐Khorassani from the Department of Chemistry and Dr. Sherry Hildreth at the Virginia Tech Mass Spectrometry Incubator (VT‐MSI), whose support was instrumental to this work. Figures were created using Biorender.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting File: mabi70111‐sup‐0001‐SuppMat.docx.

MABI-26-e00501-s001.docx (16.8MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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