The P. multocida strain used in this study was C48-1, which was purchased from the China General Microbiological Culture Collection Center (CGMCC). P. multocida was cultured at 37°C in Difco tryptic soy broth (TSB) or tryptic soy agar (TSA) (BD, Franklin Lakes, NJ, USA).

Coptisine (purity > 98%) and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) kits were purchased from Solarbio (Beijing, China). Alkaline phosphatase (AKP), succinate dehydrogenase (SDH), and lactate dehydrogenase (LDH) activity assay kits were obtained from the Nanjing Jiancheng Bioengineering Institute (Jiancheng, Nanjing, China). The Bradford protein concentration assay kit was purchased from Beyotime Biotechnology (Shanghai, China).

The minimum inhibitory concentration (MIC) was measured using the dilution method (Kowalska-Krochmal and Dudek-Wicher, 2021). The initial concentration of coptisine in the first well was 2 mg/mL, and the solution was diluted 2-fold. Finally, 100 μL of bacterial suspension (McFarland standard 0.25) was added. After culturing at 37°C for 24 h, the MIC of coptisine was identified as the well with no bacterial growth.

The half maximal inhibitory concentration (IC₅₀) was measured as previously described (Chen et al., 2021). A CCK-8 kit (Yeasen, Shanghai, China) was used to evaluate the effect of coptisine on the viability of DF-1 cells. DF-1 cells, in 100 μL of culture medium, were added at a density of 10⁴ cells/mL to 96-well plates. The cells were treated with coptisine at various concentrations for 24 h. Subsequently, the IC₅₀ was estimated according to the manufacturer’s instructions. The experiments were performed in triplicate.

A growth curve was constructed using the viable cell count method. Briefly, independent bacterial colonies were inoculated in TSB and cultured overnight with shaking. The resulting bacterial culture was then transferred to fresh TSB medium containing different concentrations of coptisine (0, 0.5 MIC, MIC, and 2 MIC) at a ratio of 1:1000 (v/v) and grown at 37°C with shaking at 200 rpm. Colony-forming units (CFUs) were measured by agar pouring inoculation, and this test was done at 2-h intervals for a total of 18 h.

The morphological characteristics of P. multocida cell membranes were observed using transmission electron microscopy (TEM). P. multocida cultured at an initial concentration of 10⁷ CFU/mL was exposed to the MIC concentration of coptisine, and the mixtures were then incubated at 37°C with shaking at 200 rpm for 4 and 8 h, respectively. Cultures without coptisine were used as controls. Cells were collected, dehydrated, embedded, and stained as previously described (Feng et al., 2016). The morphologies of the prepared samples were observed using TEM.

A suspension of P. multocida, cultured to the mid-exponential phase, was transferred to fresh TSB containing the MIC of coptisine. The initial concentration of the medium was 10⁷ CFU/mL. The mixtures were incubated at 37°C with shaking at 200 rpm, and cultures without coptisine were used as controls. Samples were collected from each tube at 0, 2, 4, 6, 8, 10, and 12 h. The activity of AKP was measured using an AKP activity assay kit.

P. multocida was cultured in TSB until it reached the mid-exponential phase. Then, the culture was incubated at 37°C with shaking at 200 rpm for 10 h. Three separate experiments were performed for each group. The suspension was centrifuged at 5000 rpm for 10 min, and the sediment of the bacterial sludge was washed three times with a 0.1 mol/L Tris-HCl buffer. Then, an equal volume of lysozyme solution was added to the bacterial sludge, incubated at 37°C for 20 min, and then immediately placed in an ice bath. The solution was mixed with 1 mL Tris-SDS buffer and centrifuged at 10,000 rpm for 15 min at 4°C. The SDH and LDH activities in the supernatants were determined according to the manufacturer’s instructions.

The P. multocida strain was cultured in TSB until it reached the mid-exponential phase. The culture was transferred to fresh TSB containing different concentrations of coptisine (0, 0.5 MIC, MIC, 2 MIC) at a ratio of 1:1000 (v/v). The culture was incubated at 37°C with shaking at 200 rpm for 10 h. A medium without coptisine was used as a control. The number of cells was adjusted to the same value, and the cells were collected by centrifugation. The precipitate was suspended with loading buffer and boiled at 100°C for 10 min. The supernatant was prepared by centrifugation at 4000 rpm for 10 min at 4°C. The supernatant was resolved with SDS-PAGE. The gel was stained with 0.05% Coomassie Brilliant Blue R-250 and de-stained to obtain soluble protein bands. The protein bands were quantified using the Gel-Pro Analyzer 4 software.

The content of genomic DNA was determined using gel electrophoresis and DAPI staining methods. P. multocida was cultured until the mid-exponential phase. The culture was adjusted to a concentration of 1×10⁶ CFU/mL using TSB. Following the addition of the MIC of coptisine, the culture was then incubated at 37°C for 30, 60, 90, and 120 min. Sterilized water was used as the control. The number of cells was adjusted to the same value, and the cells were collected by centrifugation. Genomic DNA was extracted using a DNA extraction kit (TianGen, Beijing, China) and analyzed by 1% agarose gel electrophoresis. The optical densities of the bands were analyzed using Gel-Pro Analyzer 4 software.

The DAPI assay was performed as described previously (Liu et al., 2022). First, P. multocida culture was added to the TSB medium containing the MIC of coptisine at a final concentration of 1×10⁶ CFU/mL. The cultures were kept at 37°C with shaking at 200 rpm for 10 h. The number of cells was adjusted to the same value, and they were suspended in the same volume. Subsequently, 1 μg/mL DAPI was added to the 1 mL suspension and incubated in the dark at 25°C for 1 h. The intensity of the blue DNA fluorescence was then observed under a fluorescence microscope.

P. multocida was cultured in TSB until it reached the mid-exponential phase. The culture was then transferred to fresh TSB at 1:100 (v/v). The culture was incubated at 37°C with shaking at 200 rpm. A 2 MIC concentration of coptisine was added to the culture at an OD600 of 0.6. The cultures were maintained for 2 h under similar conditions. A medium without coptisine was used as a control. Total RNA was extracted using an RNA Extraction Kit (Omega, Norcross, GA, USA). The total RNA was treated with RNase-free DNase I (Promega, Wisconsin, USA) at 37°C for 30 min to obtain the purified RNA. Purified RNA was sent to Shanghai Majorbio Biopharm Technology Co., Ltd. (Shanghai, China) for RNA-seq. All RNA samples were analyzed in triplicate.

Differentially expressed genes were verified using reverse transcription quantitative real-time PCR (RT-qPCR). Primers were designed using the Primer 6.0 software; the sequence of primers is shown in the Supplementary Material . RNA was extracted as previously described. RNA was reverse transcribed to cDNA using the PrimerScript RT Reagent Kit with a gDNA Eraser (Takara, Dalian, China). RT-qPCR was performed using SYBR Premix (Vazyme, Nanjing, China). The 16S rRNA gene was used as a reference gene. Relative quantification of gene expression was calculated using the 2^-△△Ct method.

All experiments were performed in triplicate. Student’s t-test was used to determine whether there was a significant difference (P < 0.05) using Prism software (GraphPad Software, San Diego, CA, USA).

The MIC of coptisine against C48-1 was 0.125 mg/mL, whereas methyl alcohol (12.5%) had no effect on the growth of P. multocida. The IC₅₀ of coptisine on DF-1 cells was found to be greater than 2 mg/mL, which is the concentration of its mother liquor, thus indicating its safeness to the organism. The antibacterial kinetic curves are shown in Figure 1A . The strain grown in the medium containing coptisine showed a slower growth rate than that grown in the medium without coptisine. The 2 MIC concentration of coptisine completely inhibited strain growth. This indicates that coptisine has a strong inhibitory activity on P. multocida, which occurred in a concentration-dependent manner.

Cell membrane permeability was assessed by measuring extracellular AKP activity (Liu et al., 2022). The extracellular activity of AKP in the coptisine treatment group was significantly higher than that in the control group ( Figure 1B ). This result reveals that AKP leaked into the extracellular space, indicating that coptisine increases the normal permeability of bacterial cell membranes.

The integrity of the cell membrane structure was observed using TEM. The TEM image of the untreated cells was smooth and complete, and the chromatin was homogeneously distributed in the center of the cell ( Figure 2A ). After coptisine treatment for 4 h, membrane integrity and cytoplasmic distribution were slightly disrupted ( Figure 2B ). After coptisine treatment for 8 h, the cell damage became more severe, and the cells exhibited morphological deformations and cytoplasmic vacuolation ( Figure 2C ). This result shows that coptisine destroys the structure of P. multocida.

The SDH activity of cells treated with coptisine at the MIC was 5.80 ± 0.74 U/mg protein, which was significantly lower than that of untreated cells (31.53 ± 1.27 U/mg protein; P < 0.05). In addition, LDH activity significantly decreased (P < 0.05). The values of LDH activity of cells treated with and without coptisine were 6.53 ± 0.36 and 3.51 ± 0.42 U/mg protein, respectively. After treatment with coptisine, the SDH and LDH levels decreased by 81.6% and 46.4%, respectively. This reveals that coptisine inhibits the activity of SDH and LDH, both of which are involved in the respiratory system of P. multocida.

The effects of coptisine on the soluble protein content of P. multocida are shown in Figure 3 . The protein bands of cells treated with coptisine at the MIC were significantly lower than those in the untreated group, as depicted in Figure 3 . Furthermore, as the dose of coptisine increased, the protein bands progressively decreased. The amount of soluble protein in the 0.5 MIC, MIC, and 2 MIC treatment groups was reduced by 4.5, 14.5, and 28.1%, respectively, when compared with the untreated group. The results show that coptisine inhibits soluble protein synthesis in P. multocida.

The genomic DNA content of P. multocida cells is shown in Figure 4 . The bands corresponding to genomic DNA content gradually increased over time. The bands in the control group were deeper than those in the coptisine-treated group ( Figure 4A ). The gray value was measured using gel analysis software ( Figure 4B ). The genomic DNA content of P. multocida cells treated with coptisine at MIC was significantly lower than that of the control group. This indicates that coptisine continuously inhibits the synthesis of bacterial genomic DNA. The DAPI assay results showed that the fluorescence of the coptisine group was significantly less than that of the control group ( Figure 5 ). Since the number of cells was adjusted to be the same, the inhibition of bacterial genomic DNA synthesis by coptisine is implied, which is consistent with the results obtained from the measurement of genomic DNA content.

Gene expression was compared between cells grown under normal conditions and those cultured in the presence of coptisine. The differentially expressed genes between samples were assigned according to the |log2 ratio| >1 and P ≤ 0.05 (Love et al., 2014). In total, 424 genes were upregulated, and 360 were downregulated, as shown in Figure 6A . The trends in RT-qPCR were consistent with the results observed in the RNA-seq analysis, as illustrated in Figure 6B .

According to the KEGG pathway classification, differentially expressed genes were primarily involved in amino acid metabolism, genetic information processing, and cellular processes ( Figure 7 ). The results of the pathway enrichment analysis of the differentially expressed genes are shown in Figure 8 . Downregulated genes were primarily involved in ABC transporters, quorum, and amino acid metabolism ( Figure 8A ). The upregulated genes were mainly involved in ribosome and aminoacyl-tRNA biosynthesis and RNA degradation ( Figure 8B ).