All the strains in this experiment were obtained from the strain preservation library of the Microbiology Department of the Laboratory of Qingyuan People’s Hospital. Twenty strains of extensively drug-resistant Pseudomonas aeruginosa were randomly selected from the strain preservation bank, and the specimens were taken from blood, sputum, pleural and abdominal fluid and other fluids of clinical patients in our hospital.

Sodium houttuyfonate (HARVEYBIO, 97%, high purity); Meropenem (Solarbio/Solarbio); Meropenem drug-sensitive paper tablets (Liofilchem/Liofilchem); sterile PBS solution (ECOTOP SCIENTIFIC); blood plate (Jiangmen Kailin Trading Co., Ltd.); broth medium and MH agar plate (Guangzhou Dijing Ltd.); LB liquid medium (tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L); 1% crystal violet staining solution (Solarbio/Solarbio); Trizol lysate (BioFluX); reverse transcription kit (MedChem Express); SYBR Green qPCR Master Mix (MedChem Express); 96-well plate (Beijing Xinxing Qiangsen Biotechnology Co, Ltd.); DensiCHEK Plus Electronic Turbidimeter (bioMérieux USA Inc.); essenscienELF6 mini centrifuge (Guangzhou Keqiao Experimental Technology Equipment Co, Ltd.); KDC-40 low-speed centrifuge (Anhui Zhongke Zhongjia). (Anhui Zhongke Zhongjia); desktop high-speed freezing centrifuge (Germany SIGMA1-16K); enzyme labelling instrument (Germany BMGSpectrostarNamo full-wavelength); BIOMATE160 UV spectrophotometer (Thermo Fisher Scientific); orthogonal fluorescence microscope (Japan Olympus BX53); fluorescence quantitative PCR (Bohle CFX-CONNEC).

In order to obtain a single colony, we activated the selected 20 strains of Pseudomonas aeruginosa, transferred them to the blood plate by plate streaking method, and incubated them in a microbial incubator at 37 ° C for 24 h. After 24 h of culture, single colonies were picked with a sterile disposable cotton swab, mixed in a sterile PBS solution, and the concentration of the solution was adjusted to 1.5 × 10⁸ CFU/ml. Sodium houttuynium and Meropenem were dissolved in pure water, and the concentration was adjusted to 64,000 μg/ml and 2028 μg/ml, respectively.

Screening for heteroresistant strains was performed using the Kirby-Bauer disk diffusion test. A disposable sterile cotton swab was used to spread the bacterial solution at a concentration of 1.5 × 10⁸ CFU/ml over the entire surface of the MH medium. After the liquid was absorbed, a Meropenem disk was placed in the centre of the medium and incubated in a microbiological incubator at 37°C for 24 h. We preliminarily recognized the colonies growing in the inhibition circle as heteroresistant strains by visual observation. The strains initially recognized as heterogeneous were identified as homologous to the original strain by the VITER-2 Compact fully automated microbiological analysis system. According to the literature, if Highest Inhibitory Concentration (HIC) at which the antibiotic exhibits the greatest inhibitory effect on the bacteria is higher than eight times the Highest Noninhibitory Concentration (HNIC), then the strain can be defined as a heteroresistant strain (El-Halfawy and Valvano, 2015). Four heteroresistant strains were identified, and were used in the subsequent experiments, with the numbers of strains 10,512, 10,517, 11,617, and 11,643, respectively.

The MICs of Sodium houttuyfonate and Meropenem against heteroresistant strains of bacteria was determined by micro broth dilution method. To the 96-well plate, 100 μl each of the bacterial solution with 1.5 × 10⁸ CFU/ml and the drug solution with diluted concentration were added, making the final concentration of Sodium houttuyfonate 32,000, 16,000, 8,000, 4,000, 2,000, 1,000, 500, 250, 125, 63, 32, 16 μg/ml; and the final concentration of Meropenem 10,240, 5,120, 2,560, 1,280, 640, 320, 160, 80, 40, 20, 10, 5 μg/ml. sterile LB liquid medium was set as blank control, and the suspension without any drug was negative control, mixed and incubated at constant temperature at 37°C for 20 h. After incubation, the MIC is determined as the lowest concentration of the antimicrobial agent that completely inhibits visible bacterial growth (Parvekar et al., 2020).

The MICs of Sodium houttuyfonate and Meropenem were measured by the checkerboard dilution method, and the inhibitory index of the combination was calculated to measure the effect of the combination. In the 96-well plate labelled 1–8 horizontal columns, Sodium houttuyfonate was continuously diluted at twice the rate; in the columns labelled A-H, Meropenem was diluted at twice the rate. Add 50 μl of Sodium houttuyfonate and Meropenem into each well; then add 50 μl of 1.5 × 10⁸ CFU/ml bacterial solution into each well, and finally add MH liquid medium into each well to make up to 200 μl. The concentration gradient of Sodium houttuyfonate and Meropenem was determined according to the results of the experiment; meanwhile, set up the sterile LB liquid medium in the columns of 9–12 to be the blank control, and the bacterial solution without any drug was used as the negative control. as a negative control, mixed and incubated at a constant temperature of 37°C for 24 h (Figure 1). After the end of the culture, the OD₆₀₀ of each well was measured by microplate reader, and the MIC of the drug was the smallest concentration of the drug that inhibited more than 80% of the bacteria, FICI = MIC of A drug combination/A drug alone MIC+MIC of B drug combination/B drug alone MIC; FICI≤0.5 is synergistic, 0.5 < FICI≤1 is additive, 1 < FICI≤2 is irrelevant, and FICI > 2 is antagonistic. FICI >2 is antagonistic.

According to the determined MIC values, Sodium houttuyfonate and Meropenem were diluted to 2 times MIC, 1 times MIC, ½ times MIC concentration for each strain with fresh MH liquid medium, respectively. Logarithmically grown bacterial suspension was turbidimetrically adjusted to 1.5 × 10⁸ CFU/ml with MH liquid medium. Each strain was prepared as ① 1 times MIC concentration of SH (1 × MIC SH), ② ½ times MIC concentration of SH (½ × MIC SH), ③ 1 times MIC concentration of MEM (1 × MIC MEM), ④ ½ times MIC concentration of MEM (½ × MIC MEM), ⑤ 1 times MIC concentration of SH and 1 times MIC concentration of MEM (1 × MIC SH + 1 × MIC MEM), ⑥ ½ times MIC concentration of SH and ½ times MIC concentration of MEM (½ × MIC SH + ½ × MIC MEM), and ⑦ negative control; the suspension without any drug was the negative control. Each tube was incubated with the same concentration of the final bacterial solution at 37°C in a constant temperature incubator with shaking; OD₆₀₀ was measured at 8 time points, respectively 0, 4, 8, 12, 24, 36, 48, and 72 h. The time-killing curves of the strains were plotted with the time points as the horizontal coordinates and the OD₆₀₀ as the vertical coordinates (Qu et al., 2016; Rogers et al., 2022).

The biofilm inhibition test of heterogeneous Pseudomonas aeruginosa by Sodium houttuyfonate was determined by Crystal violet staining (Masihzadeh et al., 2023). Suspensions in the logarithmic growth phase were taken and diluted to 1.5 × 10⁸ CFU/ml. LB liquid medium containing different concentrations of Sodium houttuyfonate was prepared using LB liquid medium as a solvent for Sodium houttuyfonate. To each 96-well plate, 100 μl of bacterial suspension and drug solution were added, making the final concentration of Sodium houttuyfonate 125, 250, 500, 750, 1,000, 1,500, 2,000, 4,000, 6,000, and 8,000 μg/ml. each drug concentration possessed three replicate wells, and at the same time, sterile LB liquid medium was set up as a blank control. The suspension without any drug was the negative control. After thorough mixing, the incubation was left at 37°C for 18–24 h. After the incubation, the culture medium was discarded and washed with sterile PBS to preserve the biofilm as much as possible. After drying, the biofilm was fixed with anhydrous ethanol for 15 min; after fixation, the anhydrous ethanol was discarded. The biofilm was stained with 0.1% crystal violet staining solution for 5 min; after staining, the staining solution was washed away with sterile PBS; finally, 33% acetic acid solution was added to dissolve the biofilm after air-drying. The 96-well plate was incubated at 37°C for 30 min, and the absorbance of each well was measured at 570 nm using a microplate reader.

Put a microscope slide into a 6-well plate, add 0.5 ml of suspension with 1.5 × 10⁸ CFU/ml, 0.5 ml of different concentrations of Sodium houttuyfonate, 1 ml of LB liquid medium and mix thoroughly, so that the final concentrations of Sodium houttuyfonate were 0, 250, 500, 1,000, 2,000 and 4,000 μg/ml. At the same time, set up a negative control group without the addition of Sodium houttuyfonate, and put it into a 37°C incubator for 18–24 h (Haney et al., 2021). The subsequent steps were the same as in experiment 2.8, and the stained coverslips were placed on the slides and observed using a light microscope with a magnification of 1,000 times.

Bacterial swimming motility assay was used to determine the effect of Sodium houttuyfonate on the swimming ability of heterogeneous Pseudomonas aeruginosa (Qu et al., 2016). Swimming plates (LB agar with 0.3% w/v agar) containing different concentrations of Sodium houttuyfonate (0, 250, 500, 1,000 and 2,000 μg/ml) were prepared. After the plates were dried, single colonies were inoculated in the centre of the plate using a sterile toothpick and incubated at a constant temperature of 37°C for 24 h. Swimming diameters of each group were determined and compared with the control group to determine the effect on the ability to swim. All experiments were performed in triplicate using the same bacterial strain, and the mean values of the results were calculated and included in the statistical analysis.

Each bacterial strain was set up with Sodium houttuyfonate (2,000 μg/ml) treated group and control group without Sodium houttuyfonate treatment. Total RNA was extracted from biofilm-state bacteria using TRIzol reagent, with RNase-free water serving as a blank control. The A260/A280 ratios of all RNA samples ranged between 1.9 and 2.1, confirming high purity. Equal amounts of RNA were reverse-transcribed into cDNA using a reverse transcription kit according to the manufacturer’s protocol, and stored at −20°C for subsequent use.

Real-time fluorescence quantitative PCR (RT-qPCR): The relative expression levels of genes related to biofilm formation and genes related to swimming ability were calculated according to the 2-ΔΔCt formula using SYRB Green staining method, and the homologous untreated strain of Pseudomonas aeruginosa treated with sodium ichthyocyanin at 2,000 μg/ml was used as the reference strain, and 16SrRNA gene was used as the reference gene. Calculations are made by taking the average of 3 replicate holes for each sample. The average of three wells for each sample was taken for calculation, and the RT-qPCR primers required for this experiment were designed using Primer Premier 6.0 (Supplementary Table S1). The reaction system and conditions were set up according to the instructions of the real-time fluorescence quantitative PCR kit, and the specificity of the products was determined by melting curve analysis.

Statistical analyses of data are expressed as mean ± standard error. Independent t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons between more than two groups. All statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). All statistical tests were two-sided and p < 0.05 was considered statistically significant.

Initial screening using the disk diffusion method revealed scattered colonies within the inhibition zones, suggesting potential heteroresistance (Figure 2). Turbidimetric validation in LB broth confirmed four strains with a heterogeneous inoculum effect (HIC/HNIC ≥8) (Table 1).

The MICs of Sodium houttuyfonate (SH) and Meropenem (MEM) were determined via broth microdilution in 96-well plates. SH exhibited a consistent MIC of 4,000 μg/ml across all strains, while MEM showed strain-dependent inhibitory activity (Table 2).

Checkerboard assays revealed synergistic inhibition (FICI ≤ 0.5) of SH and MEM against all four strains (Table 3). Heatmaps generated with Prism software visualized growth patterns across drug concentration gradients (Figure 3).

Time-kill curves demonstrated that SH alone (1 × MIC SH and ½ × MIC SH) achieved bactericidal effects comparable to MEM. However, combinations of SH and MEM (1 × MIC SH + 1 × MIC MEM or ½ × MIC SH + ½ × MIC MEM) exhibited enhanced efficacy, with sustained suppression of bacterial regrowth over 72 h (Figure 4). For example:

Strain 10,512: Combination therapy reduced viable counts by >1.5 × 10⁸ CFU/ml within 12 h, maintaining suppression until 72 h.

Crystal violet staining revealed dose-dependent biofilm inhibition by SH (≥250 μg/ml, p < 0.001 vs. untreated control). Notably, sub-MIC concentrations (25–125 μg/ml) transiently enhanced biofilm formation (Strain 10,512 and Strain 11,617 p < 0.05), followed by suppression at ≥250 μg/ml (Figures 5–7). This biphasic response suggests SH may initially promote bacterial attachment at low doses before exerting inhibitory effects.

Microscopic analysis of crystal violet-stained biofilms showed reduced bacterial density and structural loosening in SH-treated groups (Figure 8). Higher SH concentrations (>500 μg/ml) correlated with diminished biofilm viscosity and dispersal of bacterial clusters.

SH significantly impaired swimming motility at ≥250 μg/ml, as evidenced by reduced colony diameters on soft agar (p < 0.01 vs. control; Figures 9, 10).

qRT-PCR analysis indicated SH downregulated biofilm-related genes (pslA, pelA, algD, lasI, lasR, rhlA) and motility-associated genes (fliC, pilA, pilZ) (p < 0.01; Figures 11, 12). These findings align with observed phenotypic changes in biofilm and motility assays.

This study demonstrates that Sodium houttuyfonate synergizes with Meropenem to combat heteroresistant Pseudomonas aeruginosa by targeting both planktonic and biofilm-associated virulence. Our results highlight three critical advances: