The MIC of DAP or TOB (Beijing Solaibao Technology Co., Ltd., Beijing, China) or ethylenediaminetetraacetic acid disodium (EDTA-2Na) (Shanghai Test One Chemical Reagent Co., Ltd., Shanghai, China) against the experimental strain was determined using the method described by Belanger et al (27). The PA PAO1 wild-type strain was obtained from the Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, Singapore (28, 29). To prepare the P8 bacterial solution, 1–3 pure colonies were selected and cultured on agar plates overnight. The colonies were then further cultured with Müller-Hinton broth (Qingdao Hopebio Biotechnology Co. Ltd., Qingdao, China) at 37°C and 220 rpm for 14–16 h. The resulting bacterial solutions were centrifuged at 3,000 rpm for 15 min using a centrifuge from HERMLE Labortechnik GmbH (Germany), and any residual media components were removed. The strains were then thoroughly washed three times with phosphate buffer solution (PBS). The concentration of the bacterial solution was adjusted to 1.0 × 10⁸ CFU/mL (OD₆₀₀ = 0.1) using a spectrophotometer (UV-Visible Spectrophotometer T6, Beijing Purse General Instrument Co. Ltd., Beijing, China). DAP (Shanghai Macklin Biochemical Co. Ltd., Shanghai, China) was dissolved in dimethyl sulfoxide (DMSO) (Beijing Solarbio Science Technology Co. Ltd., Guangzhou, China), while TOB was dissolved in normal saline. Different final concentrations of DAP and TOB solutions were prepared (the DMSO solvent was diluted at least 100-fold). Approximately 95 μL of the mixture was added to each well of a 96-well cell culture dish, followed by the addition of 5 μL (OD₆₀₀ = 0.01) of the bacterial solution to each well, with three wells being repeated. In addition, a negative control group (without bacterial solution) and a positive control group (without drugs) were established. The 96-well cell culture dish was placed in a biochemical incubator (HPX-400, Shanghai Yuejin Medical Equipment Co. Ltd., Shanghai, China) at 37°C for 18 h. Finally, the MIC was determined based on the turbidity of the culture. Then, the wells where the concentration was greater than or equal to MIC were incubated at 37°C for an extra 24 h. The concentration at which there was still no visible growth was defined as the MBC (30).The experiment was repeated three times.

The following method, developed by Haney et al. (31), was used as a reference. A P8 bacterial solution was prepared and 200 μL of the bacterial solution was added to each well of a 96-well cell culture dish. The medium was changed every other day and cultured at 37°C for 72 h. Before treatment, the OD₆₀₀ value of the bacterial solution was measured using a microplate reader. The suspended bacterial solution of each well was then discarded and the BF was gently washed three times with PBS solution, leaving only the adherent growth BF. In vitro experiments were divided into the following groups: control group (same amount of medium added without drugs), DMSO group (solvent DMSO diluted 100 times, solvent only), drug group: DAP group (890 μg/mL), TOB group (5.5 μg/mL), EDTA-2Na (2 mg/mL) + TOB (5.5 μg/mL), and DAP (890 μg/mL) + TOB (5.5 μg/mL) group. EDTA-2Na served as a positive control for the experimental treatment. Different groups of 200 μL drugs were added to 96-well cell culture dishes and further cultured at 37°C for 24 h. The supernatant of each well was discarded, and the BF was gently washed three times with PBS solution and then dried at room temperature. A 96-well cell culture dish was stained with 220 μL of 1% crystal violet (100 mL, Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) at room temperature for 30 min. The dye was then poured out, the dish was gently washed three times with pure water and then left to dry (usually for 24 h). A total of 220 μL of 33% glacial acetic acid was added to the 96-well cell culture dish and dissolved at room temperature for 20 min. The OD value was measured at a wavelength of 595 nm using a microplate reader (3020, Thermo Fisher Scientific Oy, Finland).

To determine the concentration of bacteria, the method of He et al. (32) was used. After drug treatment, the supernatant of the original well was removed and the BF was washed three times with PBS solution. Then 100 μL of 0.1% Triton X-100 was added to the well and sonicated for 5 min using a JP-031S sonicator. The resulting bacterial suspension was diluted tenfold using serial dilution and spread evenly on an LB agar plate. After 24 h of incubation at 37°C, the CFU count was performed and the bacterial concentration was calculated. Finally, the results were recorded as logarithmic values with 10 as the base.

To prepare the BF carrier, a circle of 1 cm diameter was cut from a 0.3-mm thick polyvinyl chloride (PVC). This circle was soaked in 75% alcohol for one night and then rinsed three times with sterile 0.9% normal saline (NS) to obtain the BF carrier. Next, 1,000 μL of P8 solution was added to a 24-well cell culture dish. Four holes were made and the BF carrier was placed in each hole, ensuring that it was completely immersed in the bacterial solution. The dish was then cultured at 37°C for 72 h. The medium was changed every other day and surface planktonic bacteria were removed by rinsing with sterile PBS water. In vitro experiments were conducted using fine groups: control, drug group (890 μg/mL DAP), TOB group (5.5 μg/mL), EDTA-2Na (2 mg/mL) + TOB (5.5 μg/mL), and DAP + TOB group (890 μg/mL DAP + 5.5 μg/mL TOB). One thousand microliters of the respective drug solutions were added to 24-well cell culture dishes and cultured at 37°C for 24 h. After 24 h, the supernatant of each well was discarded and the BF carrier was gently washed three times with PBS solution. It was then placed in 2.5% glutaraldehyde fixative and fixed at 4°C for 12 h. After the removal of the glutaraldehyde, the BF carrier was rinsed with PBS three times for 5 min each time. The BF carrier was dehydrated through a series of ethanol concentrations (30% → 50% → 60% → 70% → 80% → 90%) for 10 min each time. It was then dehydrated twice in 100% ethanol for 10 min each time. The samples were dried in a vacuum freeze dryer for 24 h and then sprayed with gold using the ion sputtering coating method. Finally, the samples were observed and photographed under the SEM. Finally, we performed quantitative analysis of electron microscopy images using Image-Pro Plus 6.0 software (Image-Pro Plus 6.0, Media Cybernetics, USA).

Healthy and clean New Zealand rabbits, aged 3–4 months, provided by the Experimental Animal Centre of Guangxi Medical University were used in this study. One week before the experiment, each rabbit was placed in an individual cage and provided with water and antibiotic-free rabbit food (80–100 g) at a temperature of 18–25°C with air circulation and 40–70% relative humidity. The rabbits (n = 16) were randomly divided into four groups using a random number table. These groups were the control group (which received the same amount of sterile saline without drugs), the DAP group (which received 890 μg/mL), the TOB group (which received 5.5 μg/mL), and the TOB + DAP group (which received both drugs at the same concentration as the other groups). The study was approved by the Medical Ethics Committee of Guangxi Medical University, and all animal experiments were conducted following the committee’s guidelines.

To prepare the bacterial inoculation solution, the P8 bacterial solution was first prepared. This was then diluted 10-fold to a concentration of 10⁶ and set aside. For anesthesia, the rabbits were placed in a rabbit box and slowly injected intravenously with 10% chloral hydrate (5 g/50 mL) X mL (200 mg/kg). For inoculation, the rabbits were fully anesthetized and placed on an operating table in the supine position. Next, the skin of the right knee was prepared with a standard iodophor disinfectant wipe. The knee was then flexed and the patellar ligament and the lateral space between the proximal tibia and distal femur were palpated. Next, 1 mL of the inoculum was injected using a 5 mL syringe. The rabbits were fed routinely for 6 days after inoculation.

On day 7 of the experiment, two rabbits were randomly selected from each group. Synovial fluid was collected from the infected knee joint for bacterial culture and drug susceptibility testing. The rabbits in each group were injected with one of the following: saline, DAP 890 μg/mL, TOB 5.5 μg/mL, or a combination of DAP 890 μg/mL and TOB 5.5 μg/mL in a 1 mL solution. In addition, rabbits in each group received an intramuscular injection of TOB (10 mg/kg/d) for seven consecutive days to treat any systemic infections.

On day 14, the rabbits were euthanized using intravenous injection of a lethal dose (200 mg/kg) of 20% chloral hydrate (10 g/50 mL). Samples were then collected by moving the rabbit to the operating table and placing it in the supine position. The right knee joint was disinfected with iodophor and an anterior median incision of approximately 6 cm was made. The skin, subcutaneous tissue, superficial fascia, and deep fascia were incised in sequence. The distal insertion of the patellar ligament was then cut to expose the entire joint space. The results were then photographed. The exudate, debris, and prepatellar synovium in the joint cavity were collected. Additionally, we monitored rabbit body weights and measured serum alanine aminotransferase (ALT) and creatinine levels on days 1, 7, and 14 to evaluate systemic drug toxicity.

The PNA-FISH kit (AdvanDx, MA, USA) and the method described in previous literature (33) were used, with some improvements. Sample preparation: We weighed 0.15 g of flakes and rinsed them three times with a sterile PBS solution. We then added 500 μL of PBS solution and homogenized the flocs three times using a grinding machine (sample freezing grinding instrument; Guangzhou Luca Sequencing Instrument Co. Ltd., Guangzhou, China) for 1 min each time at a speed of 70 Hz/s to obtain the homogenate. Slide preparation: Slides were immersed in 75% alcohol for 30 min and then rinsed three times with a sterile PBS solution. We placed 20 μL of homogenized droplets on the slide, evenly coated it with a gun head, and dried it. Then, 10 μL droplets of the Pseudomonas aeruginosa-specific PNA probe were placed on the slide in the dark, covered with coverslips, and sealed with rubber. The slides were placed in a humidity box and hybridized in an incubator at 55°C for 60 min. We then removed the rubber and coverslip in the dark, immersed the slide in pure water at 55°C for 30 min, and dried it. We added 10 µL of DAPI staining solution for 15 min. Finally, we used a fluorescence microscope (EVOS FL Auto 2, Invitrogen by Thermo Fisher Scientific, USA) to observe and analyze the image acquisition. After imaging, we used analysis software (Image-Pro Plus 6.0, Media Cybernetics, USA) to measure the red fluorescence area in each film (to select the value of the maximum red area).

The 0.15 g flocs were washed three times with a sterile PBS solution. Then 500 µL of the sterile PBS solution was added. The flocs were homogenized three times for 1 min at a speed of 70 Hz/s using a grinding machine. The colonies of each sample were then counted using a continuous tenfold dilution method. Finally, the results were recorded as Xg (CFU/mL) based on the weight, bacterial load, and fluid volume of each sample.

The knee joint tissues were cut to a size of 5 × 5 × 2 mm and fixed in 10% formalin solution for 24 h. It was then rinsed in running water for 24 h. The tissue was then dehydrated using 85% alcohol once, 95% alcohol twice, anhydrous alcohol twice, anhydrous alcohol and xylene (1:1) once, and pure xylene twice (each for 10 min). The next step was wax immersion and embedding. The same amount of paraffin wax was added, and the tissue was immersed in a 50% paraffin-xylene solution for 10 min, followed by a 100% paraffin solution for a further 10 min. The tissue was immersed in wax at 52–56°C for 3 h and then embedded in wax at 60°C. In the sectioning step, the tissue was sectioned (thickness 5 µm), developed, and patched at 42°C for 30 min and baked at 60°C for 12 h. The tissue was then stained and destained with xylene twice (each for 5 min), 95% ethanol twice (each for 3 min), 80% ethanol once (for 3 min), distilled water once (for 1 min), H&E staining for 5 min, color separation with 1% hydrochloric acid-ethanol for 30 s, 0.5% eosin staining for 1 min, distilled water once (for 1 min), anhydrous ethanol for 5 min, and finally dried at room temperature and sealed with neutral gum. The tissues were observed under a fluorescence microscope, and images were captured using an Eclipse Ci-L microscope (Nikon, Japan).

The MIC values against PA were as follows: 890 µg/mL for DAP, 2 mg/mL for EDTA-2Na, and 2.75 µg/mL for TOB. The corresponding MBC for TOB was 5.5 µg/mL.

There was no difference in bacterial growth among the groups before treatment, as shown in Supplementary Material named “before treatment.” PA-BF was significantly reduced in the DAP monotherapy, EDTA-2Na (2 mg/mL) + TOB (5.5 μg/mL), and DAP (890 μg/mL) + TOB (5.5 μg/mL) treatment groups compared to the control and TOB monotherapy groups (P < 0.05). However, no statistical difference was observed between the EDTA-2Na+TOB and DAP+TOB combination groups. Consistent with Figure 1 , the DMSO solvent control, TOB alone, and untreated control groups showed comparable biofilm levels.

No significant reduction in viable bacterial counts within BF was observed following 24 h treatment with 5.5 μg/mL TOB alone. In contrast, treatments with DAP monotherapy, EDTA-2Na+TOB, and TOB+DAP all demonstrated significantly lower viable bacterial counts compared to the control group (P < 0.05). The DMSO solvent control, TOB monotherapy, and untreated control groups showed comparable bacterial viability ( Figure 2 ).

In Figure 3A , the control group showed a significant amount of dense BF formation on the carrier surface. They also secreted some mucilaginous substances that were tightly associated with each other, and the pathogen PA was tightly wrapped in the BF. In Figure 3B , the single TOB group showed a similar amount of BF on the carrier surface, however, the number of PA decreased slightly, and it could be considered that the planktonic cells were killed by TOB. In the DAP alone group ( Figure 3C ), the BF on the surface of the carrier reduced significantly, but there were still several bacteria attached to the carrier surface and some BF-like substances on the bacterial surface. In contrast, carriers in both the DAP+TOB group ( Figure 3D ) and EDTA-2Na+TOB group ( Figure 3E ) showed complete absence of biofilm structures, with only sparse individual bacteria observed. Quantitative electron microscopy analysis ( Figure 3F ) confirmed significant reductions in biofilm bacterial counts for the DAP monotherapy, DAP+TOB, and EDTA-2Na+TOB treatment groups compared to controls.

On day 14 of the experiment, the fluid from the affected knee joint was collected for bacterial culture and drug susceptibility testing. The results showed that PA was infected and sensitive to TOB, confirming the success of the suppurative knee joint infection model. All 16 rabbits survived throughout the experiment. In the control and DAP groups, the rabbits gradually lost their appetite and activity during the treatment. Their knee joints became swollen and painful, and had flexion contractures. In contrast, the rabbits in the TOB and TOB + DAP groups had better appetite and activity than those in the control group, with the TOB + DAP group being the best. When the knee joints were examined, the control and DAP groups had a large amount of purulent secretions and debris in the joint cavity and severe congestion of the synovium. Meanwhile, the TOB group had a moderate amount of purulent secretions and debris, with moderate synovial congestion. Finally, the TOB + DAP group had a small amount of purulent secretions and flakes with a small amount of synovial congestion, as shown in Figure 4 .

In Figures 5A, B , representing the control and the TOB groups, respectively, a significant amount of red fluorescence (PA) was surrounded by blue fluorescence (matrix), indicating the presence of BF aggregates embedded in the matrix. However, in Figures 5C, D , representing the DAP and DAP + TOB groups, respectively, the amount of BF was less than that of the control and TOB groups. The combined group had the least amount of BF. After analyzing the images using Image-Pro Plus 6.0 software, it was found that the amount of BF in the control and the TOB groups was significantly more than that in the DAP and the combined groups (P < 0.05), as shown in Figure 5E .

The results of the experiment were as follows: the colony counts of each group were expressed as Lg CFU/mL, g, and are presented as mean ± SD. The control group had a count of 7.208 ± 0.11, the DAP group had a count of 7.215 ± 0.12, the TOB group had a count of 5.945 ± 0.45, and the TOB + DAP group had a count of 3.465 ± 0.18. There was no significant difference between the DAP and the control groups (P > 0.05). However, there was a significant difference between the other groups and the control group (P < 0.05), as shown in Figure 6 .

Figures 7 , 8 show that the control and DAP groups had high levels of inflammatory cell infiltration. Moderate inflammatory cell infiltration was observed in the TOB group, and mild inflammatory cell infiltration was observed in the TOB + DAP group. The thickness of the prepatellar synovium was measured in each group (mean ± SD), as shown in Figure 9 . The control group had a thickness of 3.120 ± 0.65 mm, the DAP group had a thickness of 3.176 ± 0.56 mm, the TOB group had a thickness of 1.668 ± 0.43 mm, and the TOB + 0.890 µg/mL DAP group had a thickness of 0.922 ± 0.11 mm. There was no significant difference between the DAP and control groups (P > 0.05). However, there was a significant difference between the other groups and the control group (P < 0.05).

All rabbits survived the experimental period. Body weights ( Figure 10A ) and serum alanine aminotransferase (ALT) ( Figure 10B ) and creatinine levels ( Figure 10C ) were measured on days 1, 7, and 14. Results indicated: Weight changes: Control group showed minor weight reduction, while treatment groups remained unaffected. Biomarkers: All creatinine and ALT values fluctuated within physiologically normal ranges without significant alterations. No treatment-related systemic toxicity was observed.