C-PC was extracted from Spirulina platensis and isolated using ammonium sulphate precipitation, followed by a single-step chromatography process with DEAE Cellulose-52 and phosphate buffer (Pan et al., 2015). As a result, the high-purity C-PC (with an A₆₂₀/A₂₈₀ value of 4.36) was obtained.

PAO1 (ATCC 15692), and PA2 (collected from the First Affiliated Hospital of Wenzhou Medical College) were stored in our laboratory. The strains were grown in Luria Bertani (LB) medium at 37°C with shaking, collected by centrifugation, and diluted in the appropriate experimental medium.

RAW264.7 cells were purchased from the Institute of Biochemistry and Cell Biology Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM medium (Gibco, New York, USA) supplemented with 10% fetal bovine serum (Chinese Holly Biotechnology Ltd., Hangzhou, China).

The MIC of C-PC against PA was determined using the broth microdilution method following the guidelines of the Clinical and Laboratory Standards Institute (Gao et al., 2022). Briefly, 5 × 10⁶ colony-forming units (CFU)/mL of PA were dispensed into LB medium supplemented with 0, 16, 32, 64, 128, 256, 512, and 1024 μg/mL of C-PC or Ampicillin (AMP) (Yuanye, Shanghai, China). The plates were incubated at 37 °C for 24 h.

Briefly, 5 × 10⁶ CFU/mL of PA2 was diluted in LB medium cultured with different concentrations of C-PC (0, 64, 128, 256 and 512 μg/mL) at 37°C with continuous shaking (180 rpm). Every 2 h, the absorbance of each bacterial culture was measured at 600 nm using a microplate reader.

This experiment was performed as above in 2.4 and cultured for 12 h. For cell-free quantification of virulence factors, pyocyanin was extracted from the culture supernatant using chloroform (800 µL) at a 3:2 ratio, followed by re-extraction with 200 µL of 0.2 mol/L HCl (Yang et al., 2021). Sterilized deionized water was used in negative control. The absorbance was measured at 520 nm.

The plates were prepared according to an established protocol (Rashid and Kornberg, 2000). PA2 was cultured with C-PC (0, 64, 128, and 256 µg/mL) for 24 h. Next, 1.0 μL of PA2 culture was inoculated at the center of swimming and swarming plates. The plates were incubated at 37 °C for 12 h, and the flagellar motility was determined by measuring the diameter (mm) of the traveled cells on the surface of the agar plate (Rashmi et al., 2018).

PA2 was cultured with different concentrations of C-PC (0, 64, 128, and 256 µg/mL) for 24 h at 37 °C. After incubation, the culture medium was removed, and the tubes were washed three times with phosphate-buffered saline (PBS). The remaining biofilms were stained with 0.1% crystal violet for 15 min and then rinsed twice with water. The tubes were dried, and the biofilm-bound crystal violet was solubilized using 95% ethanol. The absorbance was measured at 595 nm (Yu et al., 2014).

For fluorescence imaging, PA2 in the logarithmic growth phase was seeded into a 24-well plate containing coverslips and incubated in LB medium with different concentrations of C-PC (0, 64, 128, and 256 µg/mL) at 37 °C. After 24 h of incubation, the medium was removed, and the formed biofilms were carefully washed three times with PBS to remove non-adherent bacteria. Subsequently, the biofilms were stained with Calcein-AM and propidium iodide (PI) (Li et al., 2015). Following two washes with PBS, the biofilms were imaged using fluorescence microscopy (Nikon, Melville, NY).

Pseudomonas quinolone signal (2-heptyl-3-hydroxy-4-quinolone, PQS) was extracted using acidified ethyl acetate (0.5% formic acid, vol/vol) and analyzed by an Accela HPLC system (Thermo Fisher Scientific,Waltham, MA, USA) (Xu et al., 2016). The assay was performed using a Zorbax Eclipse XDB-C₁₈ column (5 μm, 4.6 mm×250 mm, Agilent, Santa Clara, CA, USA). The PQS was eluted at a flow rate of 1 mL/min in an isocratic elution mode with a mobile phase consisting of 86% methanol (acidified with 1% glacial acetic acid) and 14% water (acidified with 1% glacial acetic acid). The PQS concentration in the samples was calculated by comparing the peak areas of a PQS standard (50 µg/mL, Sigma, USA) with those of PA2. The relative PQS production was normalized against the total bacterial protein content.

Total RNA was extracted and reverse transcribed into complementary DNA (cDNA), following the instructions provided in a commercial kit (Takara Bio, Inc., Dalian, China). Quantitative polymerase chain reaction (qPCR) was performed to assess the expression of QS-related genes. Supplementary Table S1 listed the primers used to amplify the reference genes. The relative differences in mRNA expression levels were calculated using the 2^-△△CT method.

To assess the cytotoxic influence of C-PC on macrophages, we conducted the the CCK-8 assay. RAW264.7 cells (1 × 10⁵ cells/mL) were seeded in 96-well plates and incubated at 37 °C overnight, and then treated with 0, 32, 64, 128, 256, and 512 μg/mL C-PC for 24 h. Then, 10 µL CCK-8 (Sangon Biotech, Shanghai, China) was added to each well, and the cells were incubated for 4 h. Afterwards, the absorbance of the cells at 450 nm was measured using a microplate reader.

RAW264.7 cells were seeded into 12-well tissue culture plates at a density of 5×10⁵ cells/well and cultured until 80-90% confluence. PA2 was prepared to a concentration of 1×10⁸ CFU/mL in DMEM without antibiotics. Cells were incubated with PA2 at three multiplicities of infection (MOIs: 25, 50, or 100) in the absence of antibiotics. After 2 or 4 h of infection, the cells were washed with PBS and lysed with 0.5% Triton-100 for 15 minutes at 37 °C. For the invasion assay, extracellular bacteria were killed by incubating with 200 µg/mL gentamicin for 1 h. The attachment and invasion levels were determined by bacterial plate count (Tang et al., 2022).

The cells were infected with PA2 (MOI = 100:1) for 4 h, followed by treatment with C-PC at concentrations of 0, 32, 64, and 128 µg/mL. The culture supernatants were collected and centrifuged at 12,000 rpm for 5 min. LDH activity was measured using an LDH cytotoxicity assay kit (Jiancheng Biotech, Nanjing, China).

All animal study protocols were reviewed and approved by the animal welfare committee of Wenzhou Medical University (approval number: SYXK2014-0052). A mouse survival assay was performed to evaluate the protective effect of C-PC against PA2 (Khayyat et al., 2021a). Five groups of healthy albino mice (5 weeks old, with similar weight) were used in the study. Two control groups received either no bacteria or an intraperitoneal (i.p.) injection of 100 µL of sterile PBS. One positive group was injected with 100 µL of untreated PA2, while two test groups were injected with 100 µL of PA2 treated with C-PC at 1/4 MIC (256 µg/mL), and 1/8 MIC (128 µg/mL), respectively. All mice were housed under standard conditions with appropriate ventilation and feeding. The survival of mice in each group was monitored daily for five days. Tissue samples were harvested for pathological analysis (Khayyat et al., 2021b).

All experiments were performed in triplicate, and the results were presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 23.0. Differences between groups were analyzed using one-way ANOVA. P < 0.05 was considered statistically significant.

The antimicrobial activity of C-PC was evaluated by the MIC microdilution method. The MICs of C-PC against PAO1 and PA2 were 512 μg/mL and 1024 μg/mL, respectively. For AMP, the MICs were 512 μg/mL and 256 μg/mL. The effect of C-PC on the growth of PA2 was assessed using growth curves at sub-MIC concentrations, with no growth inhibition observed at concentrations below 1/4 times the MIC of C-PC (Figure 1). So the sub-MIC concentration was selected to evaluate the anti-biofilm and anti-QS activity.

The inhibitory effect of C-PC on pyocyanin production was evaluated. As shown in Figure 2, pyocyanin production was significantly decreased by C-PC treatment in a concentration- dependent manner (^**P < 0.01). C-PC at a concentration of 256 µg/mL was found to have the strongest inhibitory effect on pyocyanin.

As shown in Figure 3, C-PC reduced both swimming and swarming motility (^*P < 0.05, ^**P < 0.01). 28.21, 63.34 and 68.75% decrease in swimming motility was determined in the presence of 64, 128 and 256 µg/mL C-PC, and 26.45, 63.14% decrease in swarming motility was determined in the presence of 128, 256 µg/mL C-PC against PA2. An increase in C-PC concentration led to a significant inhibitory effect on PA2 flagellar motility.

C-PC significantly reduced PA2 biofilm formation at 24 h in a dose-dependent manner (^**P < 0.01, Figure 4A). As shown in Figure 4B, untreated PA2 biofilms stained with calcein-AM emitted bright green fluorescence, indicating intact biofilms that were dense and thick. In contrast, the biofilms of the C-PC-treated group were predominantly stained with red fluorescence (PI), indicating substantial damage to the biofilm structure.

The effect of C-PC on the synthesis of signaling molecules in PA2 was assessed using HPLC analysis. As shown in Figure 5, compared with PA2, the PQS yield in cultures exposed to C-PC was significantly decreased (^**P < 0.01). C-PC at a concentration of 128 µg/mL was found to have the strongest inhibitory effect. Additionally, the expression of QS-related genes, including lasI, lasR, rhlI, rhlR, pqsA, and pqsR, were analyzed. The results showed that the expression of key genes in the PQS system, including pqsA and pqsR, was downregulated by C-PC treatment (^**P < 0.01, Figure 6A). And the expression of other key QS system genes, such as rhlI/R of the rhl system and lasI/lasR of the las system, remained unchanged.

To determine the experimental concentrations of C-PC for RAW264.7 cells in subsequent experiments, the CCK-8 experiment was used to evaluate the cytotoxic effects of C-PC. The initial concentrations of C-PC were chosen according to previously established and published protocols (Zhang et al., 2022). As depicted in Figure 7A, the viability of cells remained above 90% at concentrations of 0-128 µg/mL, indicating that C-PC was not cytotoxic to RAW264.7 cells. In the infection experiment, PA2 was able to adhere to and invade the cells. As the MOI and infection time increased, the number of PA2 bacteria attaching to and invading macrophages also increased (^*P < 0.05, ^**P < 0.01; Figures 7B, C). And compared to the PA2 group, bacteria adhesion to macrophages significantly decreased after C-PC treatment. Further analysis revealed that C-PC reduced the invasion of PA2 into RAW264.7 cells. Bacterial infection leads to membrane damage, resulting in the release of LDH from the cytoplasm into the culture medium. Compared to the control group (RAW264.7), the release of LDH was significantly higher in the PA2-infected group (^**P < 0.01). In addition, the release of LDH decreased in a concentration-dependent manner upon C-PC treatment, with no effect on macrophage proliferation at the experimental concentrations (^##P < 0.01; Figure 7D).

The in vivo protective effects of C-PC at sub-MIC concentrations against PA2 were evaluated in five mouse groups (Figure 8A). No deaths were observed in the control groups, and one mouse out of five survived in the group injected with untreated PA2. In contrast, two mice out of five survived in the 1/4 MIC group, and four mice out of five survived in the 1/8 MIC group. After 5 days of infection, histological analysis using Hematoxylin-eosin (H&E) staining revealed significantly reduced infiltration of inflammatory cells in the lung, kidney and liver of mice treated with C-PC compared to the untreated PA2 group (Figure 8B). In summary, C-PC at sub-MIC concentrations significantly protected mice from PA infection.