A total of 51 R. anatipestifer isolates were used. The isolates included ATCC11845 and 50 strains of R. anatipestifer isolated from duck farms (Supplementary Table S1). Tryptic soy broth (TSB; Huankai, Guangzhou, China) or tryptic soy agar (TSA; Huankai, Guangzhou, China) were used for the routine growth of R. anatipestifer. R. anatipestifer strains was inoculated overnight at 37°C in 2 mL of TSB with agitation at 180 rpm. IBG (99.9%) was synthesized by Guangzhou Insighter Biotechnology (Guangzhou, China). BCECF-AM was purchased from Shanghai Bioscience (Shanghai, China). DiSC₃(5) was bought from Aladdin Industrial Corporation (Shanghai, China). Propidium iodide (PI) and enhanced adenosine triphosphate (ATP) assay kits were obtained from Beyotime Biotech Inc. (Shanghai, China). Gentamicin (GEN), ethylenediamine tetraacetic acid (EDTA), and Trixon-X-100 were acquired from Sangon Biotech (Shanghai, China). Phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CA) were procured from Sangon Biotech (Shanghai, China).

The minimal inhibitory concentrations (MICs) of IBG and other antimicrobials were determined by performing the broth microdilution method in accordance with the Clinical and Laboratory Standards Institute. (2018). The MIC is the lowest concentration of IBG observed to inhibit bacterial growth after 24 h of incubation. The minimal bactericidal concentration (MBC) is the lowest concentration that reduces bacterial colonies by 99.9%. The synergistic activity between IBG and antibiotics was measured by using checkerboard assays (MacNair et al., 2018). Fractional inhibitory concentration indices (FICI) were calculated as follows: FICI = MIC   a  in combination /  MIC   a  alone + MIC   b  in combination /  MIC . b  alone

On the basis of MICs, R. anatipestifer ATCC11845 and GDH21D36 were cultured to a concentration of approximately 10⁶ CFU/mL in TSB. Different concentrations (1/4 MIC, 1/2 MIC, 1 MIC, 2 MIC, and 4 MIC) of IBG or GEN (1/4 MIC, 1/2 MIC) were added to the bacterial suspensions, and then inoculated at 37°C with agitation at 180 rpm. A tube of bacterial suspensions without the drug served as the control. All the tubes were incubated at 37°C. At 0, 1, 2, 4, 8, 12, and 24 h, 100 μL of culture was serially diluted, and the solvents were spotted onto a TSA medium. The limit of detection was 10 CFU/mL. Each experiment was performed with three replicates.

Two-week-old Cherry Valley ducks weighting 100 ± 20 g were used in this study. The ducks were provided antibacterial-free balanced feedstuff (CP FEED, Jiangsu) according to labeling and clean water. R. anatipestifer ATCC11845 was cultured in TSB and incubated at 37°C for about 16–24 h. Subsequently, bacteria were washed and resuspended in physiological saline to 10⁸ CFU/mL. Pericarditis in the R. anatipestifer-infected ducks was induced through the intraperitoneal injection of 0.5 mL of 10⁸ CFU/mL R anatipestifer ATCC11845 suspension as described previously (Qiu et al., 2016). Infected ducks received intramuscular injection 4 mg/kg b. w. Of IBG, GEN, and IBG combined with GEN with two times a day for 3 successive days (n = 6). All animal procedures were approved by the Institutional Animal Care and Use Committee of South China Agricultural University (approval number: 2022A007), and the animals were treated with consideration for their welfare and in compliance with all local and national legal requirements.

Overnight cultures of R. anatipestifer ATCC11845 were inoculated into TSB containing IBG at 1–8 μg/mL. Bacterial cells were harvested at 24 h after incubation at 37°C. Ciprofloxacin and 1% DMSO were used as a positive and negative control, respectively. Every 24 h, 30% glycerin was added to each tube with bacterial solution. The tubes were then stored at −20°C for serial passage. An MIC assay was performed through the microbroth dilution method. Experiments were performed in triplicates.

The levels of PE, PG, CA, EDTA, Trixon-X-100, LPS, and different cations (NaCl, CaCl₂, and MgCl₂) were analyzed by checkerboard assays to understand the effects of exogenous addition on the antibacterial activity of IBG against R. anatipestifer ATCC11845.

Cell membrane integrity assay was performed as a previous report (Song et al., 2020). R. anatipestifer ATCC11845 was inoculated into TSB and incubated at 37°C overnight. Bacteria were washed and resuspended in PBS to an OD₆₀₀ of 0.5. Subsequently, the fluorescent probe PI was added at a final concentration of 0.5 μmol/L. A total of 190 μL of the mixture was added to a black 96-well plates after incubation away from light at 37°C for 30 min and added with different concentrations of IBG (final concentrations of 0–16 μg/mL). Bacterial solution (100 μL) was collected from each well and transferred to a black 96-well plate after 30 min of incubation at 37°C. Fluorescence was measured at an excitation wavelength of 535 nm and emission wavelength of 615 nm.

The fluorescent probe DiSC₃(5) was used to determine the effect of IBG on the cell membrane potential (∆Ψ) of R. anatipestifer (Hamamoto et al., 2015). Overnight cultures of R. anatipestifer ATCC11845 were washed and resuspended in PBS to an OD₆₀₀ of 0.5, and the fluorescent probe DiSC₃(5) was added at a final concentration of 0.5 μmol/L. After 30 min of incubation at 37°C, 190 μL of the probe-labeled bacterial cells was collected, and 10 μL of IBG (final concentrations of 0–16 μg/mL) was added to a black 96-well plate. The mixture was mixed by blowing and suction and incubated at 37°C for 30 min. The excitation wavelength of the fluorescence spectrometer was 622 nm, and the emission wavelength was 670 nm.

Another component of the proton motive force (PMF) is the transmembrane proton gradient (∆pH), which was measured with the pH-sensitive fluorescent probe BCECF-AM (Liu et al., 2020). R. anatipestifer ATCC11845 was grown overnight at 37°C. Bacterial cells were washed and suspended in PBS until their OD₆₀₀ normalized to 0.5. A total of 190 μL of BCECF-AM was added at the final concentration of 10 μmol/L to a black 96-well plate and mixed fully with 10 μL of IBG at the final concentrations of 0, 2, 4, 8 and 16 μg/mL. The plate was incubated at 37°C for 30 min and placed in a fluorescence spectrometer with excitation and emission wavelengths of 488 and 535 nm, respectively.

The ATP levels in R. anatipestifer ATCC11845 were detected by using an enhanced ATP assay kit (Beyotime, Shanghai, China). Overnight cultured R. anatipestifer ATCC11845 cells were washed three times with PBS (pH = 7.4) and resuspended to an OD₆₀₀ of 0.5. The resuspension was added with IBG (final concentrations of 0–16 μg/mL) and incubated at 37°C for 30 min. Subsequently, cultures were centrifuged at 12,000 rpm for 5 min. Supernatants were collected to measure extracellular ATP levels. Pellets were lysed with lysozyme and centrifuged to detect intracellular ATP. ATP levels were measured by using a Hitachi F-7000 fluorescence spectrometer.

The model structure of the PgsA and PlsB proteins was obtained from the UniProt Knowledgebase (https://www.uniprot.org/uniprotkb accessed on 25 December 2023). The protein sequence was A0A126QFI4 and V4MRX6. The 2D structure of IBG was displayed using ChemDraw 20.0. Molecular docking of PgsA and PlsB proteins with IBG was performed using the LibDock protocol of Discovery Studio 2019.

GraphPad Prism 8.0 software was used for statistical analysis. All data were presented as mean ± standard deviation. One-way ANOVA was used to calculate p values between multiple groups (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

The MIC and MBC of IBG against different kinds of bacteria are shown in Table 1. IBG lacked antibacterial activity (MIC >256 μg/mL) against other Gram-negative bacteria. MIC measurements were performed on 30 R. anatipestifer isolates with various antibiotic resistance phenotypes to test the antimicrobial activity of IBG (Table 2). IBG showed better in vitro antibacterial activity against the clinical isolates than some commonly used antibiotics. The MIC range of IBG for R. anatipestifer (n = 50) was 0.5–2 μg/mL. The MIC₅₀ and MIC₉₀ of IBG were 1 μg/mL. IBG had MBCs of 1–4 μg/mL and the MBC₅₀ and MBC₉₀ of IBG of 2 μg/mL. The MICs of IBG alone and in combination with antibiotics for R. anatipestifer are listed in Table 3. The combination of IBG with GEN showed enhanced activity against R. anatipestifer with FICI values that varied from 0.38 to 0.50.

The time-killing curves of IBG combined with GEN for R. anatipestifer ATCC11845 and GDH21D24 in TSB are illustrated in Figure 2. The results showed that antibacterial activity increased with IBG concentration, indicating that the antibacterial effect of IBG on R. anatipestifer was concentration-dependent. When the concentration of IBG was less than 1×MIC, the growth of R. anatipestifer was slightly inhibited and subsequently resumed (Figures 2A, C). IBG demonstrated bactericidal activity at concentrations exceeding 2 × MIC, with no bacterial regrowth observed within 24 h. Bactericidal effects were observed when IBG and GEN were present at the concentration of 0.25×MIC and less than 1×MIC (Figures 2B, D).

The bacterial burden in lung, liver, and brain tissues of infected ducks without drug treatment was 5.59 ± 0.74 log₁₀ CFU/g. The bacterial burden in the liver of ducks treated with IBG and GEN significantly reduced (p < 0.01) compared with that in the untreated control (Figure 3). The injection of 4 mg/kg GEN with 4 mg/kg IBG significantly increased the antibacterial activity in the lung (p < 0.01) and liver (p < 0.001), reducing the bacterial load to 1.37–2.60 log₁₀ CFU/g.

In resistance studies, R. anatipestifer ATCC11845 was continuously passaged under the subinhibitory concentration of IBG. Under the pressure of IBG, the MIC of IBG for R. anatipestifer only increased two times within 30 days (Figure 4). By contrast, the MIC of CIP increased 256 times within 30 days.

R. anatipestifer ATCC 11845 was used as an indicator to explore the anti-R. anatipestifer mechanism of IBG. The fluorescence probe PI was used to measure the cell membrane integrity of R. anatipestifer after IBG treatment (Song et al., 2020). The results showed that IBG increased fluorescence intensity in a concentration-dependent manner (Figure 5A). A significant difference (p < 0.05) was found between the IBG-treated and control groups. These results indicated that in R. anatipestifer, IBG can disrupt the integrity of the cell membrane and induce membrane damage and cytoplasmic membrane dysfunction.

DiSC₃(5) was used to determine changes in membrane potential in R. anatipestifer after IBG treatment (Hamamoto et al., 2015). The fluorescence in the experimental group significantly increased (p < 0.001), and IBG significantly increased the membrane potential of R. anatipestifer (Figure 5B). Given that IBG can affect ΔΨ, BCECF-AM was used to evaluate the effect of IBG on the Δ pH of R. anatipestifer. Compared with that of the control group, the membrane potential of the IBG group had significantly reduced (p < 0.001) in a concentration-dependent manner (Figure 5C). Given that PMF disruption affects cellular ATP (Vahidi et al., 2016), intracellular and extracellular ATP levels were measured. IBG decreased intracellular ATP levels and increased extracellular ATP levels (Figure 5D). Next, investigated the effect of major cytoplasmic membrane components on the activity of IBG against R. anatipestifer ATCC 11845 under exogenous addition was investigated. The exogenous addition of bacterial phospholipids (including PG and CA) inhibited IBG activity in a dose-dependent manner (Figure 5E). The proteins PgsA and PlsB play a crucial role in the synthesis of PG and CA (Li et al., 2016). To investigate the binding interactions between IBG and these proteins, molecular docking was conducted. The results demonstrated a favorable affinity between IBG and PgsA and PlsB, as indicated by LibDockScores of 104.70 and 77.65, respectively. Additionally, the molecular docking analysis revealed potential interactions between IBG and the proteins PgsA and PlsB. In the case of the PgsA protein, the binding sites of IBG were found to contain potentially critical active residues, including TYR171, SER124, VAL121, VAL123, LYS130, ASP71, VAL75, LYS72, LEU79, ILE99, and ILE98 (Figures 6A, B). For the PlsB protein, potentially critical active residues include LEU220, LYS219, GLU368, LEU410, LYS487, GLU488, TRP486, and ARG495 (Figures 6C, D).