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. 2026 Jun 16;26:758. doi: 10.1186/s12866-026-05247-6

2’’-O-galloylhyperin as a novel adjuvant to reduce imipenem resistance in multidrug-resistant Pseudomonas aeruginosa: transcriptome-based mechanism of iron homeostasis disruption

Lin Zheng 1,#, Ge-Jin Lu 2,#, Zi-xian Wang 2, Xue-Jun Guo 2, Zhen Wang 1, Zhi-Ying Shao 1, Ye-tao Wang 1, Hua-Bin Jiang 1, Shu-Tian Chen 1, Feng-Yi Wang 2,3, Jing Li 4, Yuan-Qing Lin 4, Si-Si Zheng 4, Guang-Hui Lu 1,✉, Jun-Feng Qi 1,✉, Ling-Wei Zhu 2,✉
PMCID: PMC13501639  PMID: 42298398

Abstract

Imipenem, a critical antibiotic for treating multidrug-resistant Pseudomonas aeruginosa, now faces severe resistance from this pathogen. This study investigates the synergistic effects and underlying mechanisms of the combination of 2’’-O-galloylhyperin with imipenem against P. aeruginosa. 2’’-O-galloylhyperin showed no significant effect on the growth curve of strain 18102011 and its transconjugant D2011, while reducing the MIC of imipenem against this strain by 4-fold (FICI ≤ 0.5, synergy). This compound upregulates the citrate synthase gene gltA, and downregulates the aconitase gene acnA, enhancing citric acid synthesis while inhibiting its dehydration to cis-aconitase acid. Citric acid chelates Fe2+/Fe3+, reducing iron bioavailability, disrupting electron transfer, and increasing intracellular ROS levels in P. aeruginosa 18,102,011. Additionally, 2’’-O-galloylhyperin reduces the activity of the carbapenemase KPC-2. These findings highlight its potential as an adjuvant to enhance imipenem efficacy against P. aeruginosa infections.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-05247-6.

Keywords: 2’’-O-galloylhyperin, Pseudomonas aeruginosa, Imipenem, Oxidative damage

Introduction

Pseudomonas aeruginosa is a critical pathogen in nosocomial settings, especially in intensive care units (ICUs), where it causes severe, often fatal infections due to its biofilm formation and rapid acquisition of antibiotic resistance [1–3]. The overuse of broad-spectrum antibiotics has led to the emergence of strains resistant to last-line carbapenems, primarily mediated by carbapenemase production [4]. The emergence of pan-drug resistant strains, unresponsive to even last-resort agents like colistin, presents a profound therapeutic challenge and a major global health crisis that threatens modern medicine.

In the search for novel therapeutic strategies, natural compounds have garnered significant interest. 2’’-O-galloylhyperin, a primary bioactive flavonoid from Pyrola species, possesses significant antioxidant and anti-inflammatory properties due to its galloyl-pyrogallol structure [5, 6]. Its structural analog, quercetin, is known to inhibit biofilm formation in pathogens like Streptococcus mutans, P. aeruginosa, and Salmonella Typhimurium [7–9]. Despite this potential, the ability of 2’’-O-galloylhyperin to reverse or reduce carbapenem resistance in P. aeruginosa has not been investigated. Therefore, this study aims to evaluate the efficacy of this compound in enhancing imipenem against P. aeruginosa and to elucidate its underlying molecular mechanisms.

Materials and methods

Strains and imipenem synergist screening

P. aeruginosa strain18102011 was isolated from a bile specimen of a burn patient. The minimum inhibitory concentrations (MICs) of imipenem against strain 18,102,011 and its transconjugant D2011 were determined by the broth dilution method, and both were 4,096 µg/mL (The information of strains were listed in Table S1). In addition to imipenem, both D2011 and 18,102,011 exhibited resistance to meropenem, ceftazidime-avibactam, fosfomycin, ampicillin, piperacillin, and chloramphenicol, as determined using the BD Phoenix-100 system and E-test. Furthermore, the parental strain 18,102,011 was resistant to all other 16 antimicrobial agents tested by the BD Phoenix-100 system and E-test, including amikacin, trimethoprim-sulfamethoxazole, ciprofloxacin, and colistin. Based on its overall antimicrobial susceptibility profile, this isolate was identified as pan-drug resistant P. aeruginosa, and its imipenem resistance was horizontally transferred via plasmids [10]. Two plasmids were identified in this strain: the IncpRBL16 mega-plasmid pP2011-1 carrying blaVIM−2 and the IncP6 plasmid pP2011-2 carrying blaKPC−2 [10]. A single colony of strain 18102011 was inoculated into 2 mL of Mueller-Hinton (MH) broth and incubated with shaking at 37 ℃ for 18 h. The culture was then diluted 1:100 in fresh MH broth and incubated with shaking until the logarithmic growth phase was reached. The bacterial suspension was adjusted to 1 × 106 CFU/mL in MH broth, and aliquots were transferred into sterile 96-well plates (the final bacterial concentration was 5 × 105 CFU/mL). A library of medicinal food homology compounds (CAS: HY-CPK-15885) was screened at a fixed concentration of 1 mM. Imipenem was added to create a concentration gradient (0 to 1,024 µg/mL) across the wells.

Growth controls (bacteria without test compounds) and sterility controls (media only) were included in each assay. Plates were incubated statically at 37 ℃ for 18 h, and the optical density (OD) at 600 nm was measured. The fractional inhibitory concentration index (FICI) was calculated using the Equation [11]:

graphic file with name d33e386.gif

“a” represents the MIC of imipenem in combination; “b” represents the MIC of the compound in combination; “c” represents the MIC of the imipenem alone; and “d” represents the MIC of the compound alone. Synergy was defined as FICI ≤ 0.5, no interaction as 0.5 < FICI < 4, and antagonism as FICI > 4.

Growth curves of strain 18102011 and D2011 under treatment with 2’’-O-galloylhyperin

Single colonies of strain 18102011 and its transconjugant D2011 were inoculated separately into MH broth and cultured at 37 ℃ for 16 h. The cultures were then sub-cultured at a 1:50 ratio in fresh MH broth and incubated with shaking at 37 ℃ until the OD600 reached 0.1. The bacterial suspensions were aliquoted into sterile conical flasks and treated with gradient concentrations of 2’’-O-galloylhyperin (final concentrations: 0 µg/mL, 4 µg/mL, 8 µg/mL, 16 µg/mL, and 32 µg/mL). A solvent control group treated with dimethyl sulfoxide (DMSO) alone (final concentration: 32 µg/mL). All groups were incubated continuously, and 500 µL of culture was collected hourly from each group to measure OD600 using a microplate reader. Three biological replicates for the entire experiment. Experimental data were analyzed and visualized using the ggplot2 package in R software to plot growth curves, and to investigate the effect of 2’’-O-galloylhyperin on the growth of strain 18102011 and its transconjugant D2011. Growth curves were plotted with OD600 values on the y-axis and time on the x-axis.

Time-kill curves of strain 18102011 and D2011 following treatment with 2’’-O-galloylhyperin and imipenem

Strain 18102011 and its transconjugant D2011 were diluted with MH broth to a final concentration of 105 CFU/mL. The suspensions were evenly distributed into sterile 96-well culture plates, with 50 µL of bacterial suspension per well. The following control and treatment groups were included: (1) Strain without imipenem or 2’’-O-galloylhyperin; (2) Strain with 32 µg/mL DMSO; (3) MH broth supplemented with imipenem at 1,024 µg/mL or 2,048 µg/mL; (4) MH broth supplemented with 2’’-O-galloylhyperin at 8 µg/mL or 16 µg/mL; (5) MH broth supplemented with 2’’-O-galloylhyperin + imipenem (e.g., 8 µg/mL + 1,024 µg/mL, 16 µg/mL + 2,048 µg/mL). Bacterial counts were determined at sequential time points (0, 2, 4, 5, 7 and 8 h).

Samples from different groups were collected and serially diluted in MH broth to 10− 1 through 10− 9. The diluted bacterial solution was evenly spread on the surface of MH agar plates, which were then inverted and incubated at 37 ℃ for 18 h for colony counting. Three biological replicates for the entire experiment. Statistical analysis and visualization of experimental data were performed using the ggplot2 package in R software to plot time-kill curves, and to investigate the antibacterial effects of 2’’-O-galloylhyperin in combination with imipenem against strain 18,102,011 and its transconjugant D2011. Time-kill curves were plotted with time on the x-axis and bacterial count on the y-axis.

Nitrocefin hydrolysis

Nitrocefin is a chromogenic cephalosporin that undergoes a distinct color change from yellow to red upon hydrolysis by β-lactamases, including carbapenemases. This assay was employed to evaluate the inhibitory effects of the tested compounds on β-lactamase activity by quantitatively monitoring this colorimetric shift [12]. Details are provided below:

Strains 18102011 and D2011 were cultured in MH broth and adjusted to final concentration of 1 × 108 CFU/mL. Cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. The cell suspension was then sonicated in an ice bath (using a cycle of 5 s pulse on and 5 s pulse off at 200 W power) until lysis was complete, as confirmed by microscopic examination. The resulting lysate was clarified by centrifugation at 10,000 x g for 2 min at 4 ℃, and the collected supernatant (containing the crude enzyme extract) was kept on ice for immediate use in the subsequent assay. The reaction was set up in a sterile 96-well microplate. Each well contained 50 µL of the crude enzyme extract and 135 µL of PBS containing the appropriate drugs to achieve the desired final concentrations. The following experimental groups were established:

(1) negative control (100% enzyme activity): enzyme extract + PBS (no inhibitor); (2) positive control (inhibition control): enzyme extract + avibactam (final conc. 50 µg/mL); (3) test groups: enzyme extract + imipenem (final conc. 0, 1, 2, 4, 8, 16, 32, 64, 256, and 1024 µg/mL); enzyme extract + 2’’-O-galloylhyperin (final conc. 0, 2, 4, 8, 32, 64, and 128 µg/mL); enzyme extract + imipenem + 2’’-O-galloylhyperin in a checkerboard assay; (4) blank control: PBS + nitrocefin (to account for background absorbance); (5) substrate control: MH broth + nitrocefin (optional, to check for non-enzymatic hydrolysis).

The plate was pre-incubated at 37 ℃ for 3 min. The reaction was initiated by adding 75 µL of nitrocefin solution (final concentration 75 µg/mL) to each well, bringing the total reaction volume to 200 µL. After thorough mixing, the plate was incubated at 37 ℃ for 30 min in the dark. The absorbance at OD₄₉₀ was then measured using a microplate reader. To further assess the inhibitory spectrum of 2’’-O-galloylhyperin, the assay was repeated using a panel of clinically relevant carbapenemase-producing strains: P. aeruginosa 18,083,286 (blaIMP−1), Acinetobacter baumannii 3011 (blaOXA), Klebsiella oxytoca 3428 (blaNDM−1), and Klebsiella pneumoniae 2445 (blaKPC−2) (The information of strains were listed in Table S1). Three biological replicates for the entire experiment.

RNA preparation and transcriptome sequencing

To ensure biological reproducibility, three independent overnight cultures of P. aeruginosa 18,102,011 were prepared. On the following day, each culture was diluted separately in fresh MH broth to final density of 1 × 10⁸ CFU/mL. To ensure sufficient bacterial lysis and adequate RNA yield for subsequent transcriptome analysis, the final concentration of imipenem working solution was adjusted from 1,024 µg/mL to 512 µg/mL. Aliquots (500 µL each) of the bacterial suspension transferred to centrifuge tubes and assigned to the following treatment groups:

(1) Negative control group: treated with an equivalent volume of MH broth; (2) solvent control group: treated with 32 µg/mL DMSO; (3) imipenem treatment group: treated with imipenem at a final concentration of 512 µg/mL; (4) 2’’-O-galloylhyperin treatment group: treated with 2’’-O-galloylhyperin at a final concentration of 8 µg/mL; (5) combination treatment group: treated with imipenem (512 µg/mL) + 2’’-O-galloylhyperin (8 µg/mL).

All tubes were incubated statically at 37 °C for 7 h. After incubation, cells were harvested by centrifugation at 10,000 × g for 2 min at 4 °C. The supernatant was discarded, and cell pellets were resuspended in 100 µL of TE buffer containing lysozyme. The suspension was then incubated for 5 min at room temperature to facilitate cell wall lysis. Total RNA was extracted using the RNAprep Pure Cell/Bacteria Kit according to the manufacturer’s protocol. Ribosomal RNA (rRNA) was depleted to enrich mRNA. Sequencing libraries were then constructed from the enriched mRNA. Paired-end sequencing was performed on an Illumina NovaSeq platform. Three biological replicates were sequenced for each treatment group.

Raw sequencing reads were quality-filtered and mapped to the reference genomes of P. aeruginosa PA7 chromosome (GenBank: GCA_000017205.1), its plasmids pP2011-1 (GenBank: CP116229) and pP2011-2 (GenBank: CP116230) using Bowtie2. Read counts per gene were determined using HTSeq v0.6.1. Differential expression analysis was performed using the DESeq2 R package (v1.38.3). The following pairwise comparisons were conducted: (1) Imipenem treatment group vs. negative control group; (2) 2’’-O-galloylhyperin treatment group vs. negative control group; and (3) Combination treatment group vs. negative control group. For each gene, the log2 fold change (log2FoldChange) in expression was calculated as:

graphic file with name d33e497.gif

The resulting p-values were adjusted for multiple testing using the Benjamini-Hochberg method to control the false discovery rate (FDR). Genes with an adjusted p-value (FDR) < 0.05 and |log2FoldChange| > 1 were defined as significantly differentially expressed genes (DEGs). DEGs were visualized using volcano plots and hierarchical clustering (H-cluster) analysis.

GO function annotation

Gene Ontology (GO) enrichment analysis of DEGs was performed using the Goseq R package, with correction for gene length bias. GO terms with a corrected P-value less than 0.05 were considered significantly enriched. GO enrichment analysis was conducted for DEGs from P. aeruginosa 18102011 in the combination treatment (2’’-O-galloylhyperin + imipenem) vs imipenem treatment group. From this analysis, the top five significantly enriched GO terms were selected as the main nodes for further visualization. Subsequently, directed acyclic graphs (DAGs) were constructed for each of the three GO categories. Using the top five terms as central nodes, parent and child terms were included to illustrate the hierarchical relationships within the GO structure.

KEGG function annotation and key genes screening

The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for interpreting high-level functions of the biological system, such as the cell, the organism, and ecosystem, from molecular-level data, particularly large-scale datasets generated by genome sequencing and other high-throughput approaches (https://www.genome.jp/kegg/) [13–15]. KEGG pathway enrichment analysis of DEGs was performed using KOBAS software [16].

Protein-protein interaction (PPI) analysis of DEGs was performed based on the STRING database, which integrates known and predicted interactions [17]. Cytoscape software was used to identify potential gene modules (clusters, subnetworks) within the PPI network and to perform functional enrichment for the identification of key genes [18].

Molecular docking of 2’’-O-galloylhyperin with key proteins in significantly enriched KEGG pathways

The crystal structures of key proteins from the significantly enriched KEGG pathways of P. aeruginosa were obtained from the RCSB.PDB database (https://www.rcsb.org) [19]. The ligand structure file of 2’’-O-galloylhyperin was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) [20]. Molecular docking between the protein and 2’’-O-galloylhyperin was performed using CB-Dock2 (https://cadd.labshare.cn/cb-dock2/php/index.php) [21, 22]. Protein structures in PDB format and the 3D structure of 2’’-O-galloylhyperin in SDF format were uploaded. Blind docking was performed with other parameters set to default. CB-Dock2 automatically removed hydrogen atoms and bound ligands, and five docking runs were conducted to identify the optimal binding pocket.

Oxidoreductase, ROS and iron homeostasis assays

To assess the effects of 2’’-O-galloylhyperin on the oxidative stress responses and iron homeostasis in P. aeruginosa, the activities of key antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR)), intracellular Reactive Oxygen Species (ROS) levels, and iron ion concentrations were measured using the following protocols.

P. aeruginosa was cultured to the mid-logarithmic phase (108 CFU/mL) and harvested. Cells were lysed by ultrasonication on ice (200 W, cycles of 5 s on/5 s off) until complete lysis was confirmed. The lysate was centrifuged at 10,000 x g for 15 min at 4 °C, and the supernatant was collected. The activities of total antioxidant capacity (T-AOC), SOD, CAT, and GR were measured using commercial kits according to the manufacturer’s protocols. Absorbance was measured at the wavelength specified for each respective kit, and enzyme activities were calculated using the provided formulas. All procedures were performed on ice or at 4 °C unless otherwise indicated for incubation steps.

Intracellular ROS levels were quantified using a Reactive Oxygen Species Assay Kit. Bacteria treated with 2’’-O-galloylhyperin for 6 h were collected, washed with PBS, and incubated with 10 µmol/L DCFH-DA at 37 °C for 20 min in the dark. Cells were then washed thoroughly with PBS to remove excess probe. Fluorescence was measured at excitation/emission = 488/522 nm. ROS levels were expressed relative to the untreated control group.

Iron homeostasis was evaluated using three complementary methods: (1) Total iron content: quantified using a Prussian blue-based kit. The reaction mixture was incubated at 50 °C for 20 min, centrifuged, and the absorbance of the supernatant was measured at 700 nm; (2) Fe²⁺/Fe³⁺ ratio: determined by the 1,10-phenanthroline method. Samples were mixed with detection reagents, incubated at 37 °C for 30 min in the dark, and absorbance was measured at 510 nm (for Fe²⁺) and 364 nm (for Fe³⁺); (3) Siderophore activity: assessed using the Chrome Azurol S (CAS) assay. The culture supernatant was mixed with an equal volume of CAS detection solution. After 30 min of incubation, the reduction in absorbance at 600 nm (reflecting dissociation of the CAS-Fe³⁺ complex) was measured, with lower absorbance values indicating higher siderophore activity. All assays were performed in triplicate, and data are presented as mean ± standard deviation.

Results

Growth curves

2’’-O-galloylhyperin didn’t significantly inhibit the growth of P. aeruginosa 18102011 or its transconjugant D2011 (Fig. 1). Following the addition of 2’’-O-galloylhyperin, the growth curve of strain 18102011 was similar to that of the untreated control, with both reaching the plateau at 3 h. Only a slight and statistically non-significant growth inhibition was observed within the first hour. The bacteria entered the logarithmic phase after 2 h, consistent with the untreated group.

Fig. 1.

Fig. 1

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Growth and time-kill curve of P. aeruginosa 18102011 and transconjugant D 2011. Note: HyG, 2’’-O-galloylhyperin; IMP, imipenem. Growth curves of P. aeruginosa 18102011 and its transconjugant D2011. The x-axis shows incubation time (h), and the y-axis represents the bacterial optical density (OD600).Time-kill curves of P. aeruginosa 18102011 and its transconjugant D2011. The x-axis shows incubation time (h), and the y-axis represents bacterial count (CFU/mL)

In comparison, strain D2011 exhibited a slower growth rate, entering the logarithmic phase at 6 h and reaching stationary phase at 8 h. The inhibitory effect of 2’’-O-galloylhyperin against strains 18102011 and D2011 showed no significant concentration dependence over the range of 4–32 µg/mL.

Time-kill curves

The growth profile indicated that 2’’-O-galloylhyperin alone didn’t inhibit the growth of strain 18,102,011 or D2011 (Fig. 1). To further explore the potential synergistic interaction, time-kill assays were performed to assess the bactericidal effect of 2’’-O-galloylhyperin combined with imipenem (Fig. 1). Neither 2’’-O-galloylhyperin (8 µg/mL) nor imipenem (1,024 µg/mL) alone displayed inhibitory activity against the tested strains (P. aeruginosa 18102011 and its transconjugant D2011) (Fig. 1). However, the combined treatment with 2’’-O-galloylhyperin (8 µg/mL) and imipenem (1,024 µg/mL) produced a significant inhibitory effect that persisted for at least 4 h.

The results indicate that although 2’’-O-galloylhyperin alone does not suppress bacterial growth, it effectively restores the bactericidal activity of imipenem against both strain 18102011 and D2011.

Effect of 2’’-O-galloylhyperin on β-lactamase activity

The inhibitory effects of 2’’-O-galloylhyperin and avibactam against various carbapenemases were evaluated using nitrocefin-based assays, with activity monitored by changes in OD values (Table S1; Fig. 2). Results were strain-specific and revealed diverse interaction patterns between the two compounds:

Fig. 2.

Fig. 2

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Effect of 2’’-O-galloylhyperin and avibactam on enzyme activity in different types of bacteria. Note: B: bacterial lysis supernatant; B + CAZ-AVI: bacterial lysis supernatant + avibactam (50 µg/mL); B + CAZ-AVI + HyG: bacterial lysis supernatant + avibactam (50 µg/mL) + 2’’-O-galloylhyperin (8 µg/mL); : bacterial lysis supernatant + 2’’-O-galloylhyperin (8 µg/mL). One-way ANOVA indicated that differences between treatment groups were statistically significant (p < 0.05)

Since strain D2011 was a transconjugant of P. aeruginosa 18102011 and carried the same carbapenemases, this transconjugant was used for carbapenemase activity assays. In the transconjugant D2011 (co-harboring blaKPC−2 and blaVIM−2), 2’’-O-galloylhyperin and avibactam inhibited KPC-2 and/or VIM-2 activity, as indicated by significantly lower OD values compared to the untreated control (MIC of strains were listed in Table S2). Although avibactam demonstrated superior efficacy, its combination with 2’’-O-galloylhyperin resulted in weaker inhibition than avibactam alone, suggesting potential interference; In P. aeruginosa 18083286 (carrying blaIMP−1), neither compound inhibited IMP-1 activity, increased OD values confirmed this result; In A. baumannii 3011 (carrying blaOXA), both compounds individually strongly inhibited OXA activity. However, their combination unexpectedly eliminated the inhibitory effect, indicating a potential antagonistic interaction; In K. oxytoca 3428 (carrying blaNDM−1), neither compound significantly inhibited NDM-1 activity; In K. pneumoniae 2445 (carrying blaKPC−2), both 2’’-O-galloylhyperin and avibactam significantly inhibited KPC-2 activity. However, their combination failed to show synergy.

Effect of 2’’-O-galloylhyperin on the transcriptional level of strain 18102011

After initial data filtering and quality control checks (which included assessing sequencing error rates and GC content distribution), we obtained high-quality clean reads. For the 15 drug-treated samples of strain 18102011, each yielded over 1.1 Gb of clean Data, with the Q30 score exceeding 92% (Table S3). Overall gene expression remained stable across the various treatments, and all samples met the criteria for screening DEGs.

Figure 3 presents volcano plots illustrating DEG distributions in strain 18,102,011 under imipenem alone and combination. The x-axis represents the log2 fold-change (combination vs. imipenem alone), and the y-axis shows -log10(p-value). Red and green dots denote up- and down-regulated genes, respectively. Compared to imipenem alone, it up-regulated 1,030 genes, down-regulated 990 genes, and showed no significant change in 4,213 genes. DEGs were located on both chromosomes and plasmids.

Fig. 3.

Fig. 3

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Functional enrichment analysis of DEGs between the combination treatment group and the imipenem alone group. Note: a volcano plot illustrating transcriptional differences between the combination treatment group and the imipenem alone group; b Heatmap displaying gene expression patterns across all samples, where the horizontal axis represents sample names a1-a3: P. aeruginosa 18102011 untreated; b1-b3: DMSO (8 µg/mL); c1-c3: 2’’-O-galloylhyperin (8 µg/mL); d1-d3: imipenem (512 µg/mL); e1-e3: combination of imipenem (512 µg/mL) + 2’’-O-galloylhyperin (8 µg/mL) and the vertical axis represents log2-transformed gene expression values; c GO enrichment bubble plot for DEGs, with the x-axis indicating the rich factor of DEGs associated with a specific GO term and the y-axis listing the significant GO terms (p < 0.05); d DAG illustrating the hierarchical relationships of enriched GO terms, where the top five significant GO terms (p < 0.05) were selected as main nodes, with associated child terms connected via hierarchical edges. The color intensity of the nodes corresponds to the enrichment significance level (-log₁₀(p-value))

H-cluster analysis of the DEGs grouped all differentially expressed genes into four distinct clusters (Fig. 3). Cluster 1 and 3 showed stable expression across all groups, with no significant differences versus the untreated control. In contrast, Clusters 2 and 4 showed marked variations. Genes in Cluster 2 were significantly down-regulated in all treatment groups (DMSO, 2’’-O-galloylhyperin, imipenem, and combination) compared to the untreated control. Cluster 4 comprised 85 genes, 84 of which were chromosomal and primarily encoded electron transport chain-associated proteins. Bioinformatics analysis revealed significant down-regulation of genes encoding key components (cytochrome C, FAD-binding domain of oxidoreductase, NADPH-dependent FMN reductase, and the 4Fe-4 S iron-sulfur cluster domain) (Table S4). These findings suggest the antibacterial effect may involve disruption of the electron transport chain via transcriptional suppression.

We compared the expression of virulence factors (based on the VFDB database) and resistance genes (based on the CARD database) in P. aeruginosa 18102011 after treatment with imipenem alone or combination. Data visualization was performed using R packages “ggpubr” and “ggplot2”, and statistical significance was assessed using the t-test (see supplement Fig. 1). Compared to imipenem alone, the combination significantly suppressed the expression of multiple P. aeruginosa virulence-related genes, including genes encoding functional amyloid proteins, exolysin, secretion systems, and toxins. In contrast, genes associated with lipopolysaccharide (LPS), flagella assembly, and type IV pili were generally upregulated. Notably, the combination treatment also led to a global upregulation of antibiotic resistance-related genes, with highly expressed genes showing significant enrichment in the KEGG DNA repair pathway.

Oxidoreductase-electron transfer activity GO enrichment analysis

GO enrichment analysis revealed that the combination induced significant gene expression changes in P. aeruginosa 18102011 compared to imipenem alone. At the biological process (BP) level, 231 DEGs were identified, with 33 DEGs at the cellular component (CC) level and 160 DEGs at the molecular function (MF) level. The most pronounced changes were observed at the MF level.

A scatter plot of the top 30 MF terms demonstrated that the combination treatment significantly downregulated electron carrier activity (GO:0009055), a subcategory of oxidoreductase activity (Fig. 3). To validate functional implications, we assessed oxidative stress and iron metabolism (Fig. 4). Compared to imipenem alone, the combination did not alter antioxidant activities (SOD, CAT, GR) or total antioxidant capacity (T-AOC) in P. aeruginosa 18,102,011. However, ROS levels increased, consistent with disrupted electron transport. Siderophore production (CAS assay) and ferric ion (Fe³⁺)-reducing capacity remained unaffected, whereas the relative levels of Fe²⁺ and Fe³⁺ both declined.

Fig. 4.

Fig. 4

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Violin plots depicting the results of oxidoreductase activity, ROS levels, and iron homeostasis-related assays in P. aeruginosa strain 18102011 under different treatments. Note: FRAP, Ferric reducing antioxidant power. ND, control (no drug). DMSO, DMSO (8 µg/mL). HyG, 2’’-O-galloylhyperin (8 µg/mL). IMP, imipenem (512 µg/mL). IMP + HyG, Combination treament (imipenem, 512 µg/mL + 2’’-O-galloylhyperin, 8 µg/mL)

Propanoate metabolism and TCA cycle KEGG pathways

To investigate the mechanism of action of the combination, we first performed pathway enrichment analysis on the DEGs. The results revealed that the addition of 2’’-O-galloylhyperin, compared to imipenem treatment alone, significantly affected the citrate cycle (TCA cycle) and propanoate metabolism pathways (Fig. 5). Therefore, we focused on these two pathways for further analysis. To elucidate the interactions among the DEGs in these pathways, we constructed a PPI network using STRING database and visualized it with Cytoscape (Fig. 5). Using node degree centrality (degree ≥ 25), we identified seven hub genes: for the TCA cycle, these were citrate synthase gene gltA, 2-oxoglutarate dehydrogenase E1 component gene sucA, pyruvate carboxylase subunit B gene pycB, and 2-oxoglutarate carboxylase small subunit gene cfiB; for the propanoate metabolism pathway, they were acetyl-coenzyme A synthetase 1 gene acsA1, phosphate acetyltransferase gene pta, and pyruvate dehydrogenase E1 component subunit alpha gene pdhA .

Fig. 5.

Fig. 5

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Molecular docking of 2’’-O-galloylhyperin with key proteins in propanoate metabolism and TCA cycle KEGG pathways. Note: a x-axis: ratio of differential genes annotated to KEGG pathways; y-axis: KEGG pathways. KEGG pathway bubble chart of DEGs in the 2’’-O-galloylhyperin and imipenem combined group and imipenem alone; b core regulatory and core genes of strain 18102011’s propanoate metabolism and TCA cycle; c. the molecular docking between core genes (propanoate metabolism/TCA cycle) and 2’’-O-galloylhyperin: (1) gltA and 2’’-O-galloylhyperin; (2) pycB and 2’’-O-galloylhyperin; (3) sucA and 2’’-O-galloylhyperin; (4) acsA1 and 2’’-O-galloylhyperin; (5) cfiB and 2’’-O-galloylhyperin; (6) pta and 2’’-O-galloylhyperin

Compared to the strain 18,102,011 treated with imipenem alone, the combination significantly altered the expression of core genes in key metabolic pathways. In the TCA cycle pathway, the expression levels of gltA and sucA were significantly up-regulated, whereas those of pycB and cfiB were significantly down-regulated. In the propanoate metabolism pathway, the expression of acsA1 was significantly up-regulated, while the expression of pta and pdhA was significantly down-regulated (Table S5).

To investigate the mechanism of action of 2’’-O-galloylhyperin, we employed molecular docking analysis to evaluate its interactions with the core proteins (AcsA1, PycB, Pta, GltA, CfiB, SucA, and PdhA). Using the CB-Dock2 platform, docking calculations were conducted for each protein-ligand pair with five replicates per combination. The optimal binding conformation for each pair was selected based on binding energy. The results revealed the following binding affinities (kcal·mol⁻¹) for 2’’-O-galloylhyperin with each core protein: AcsA1 (-10.4), PycB (-9.6), Pta (-9.3), GltA (-9.2), CfiB (-8.9), SucA (-8.5), and PdhA (-8.3). To visualize the most stable interactions, the top five protein-ligand complexes (ranked by binding energy) are shown in Fig. 5. The binding models illustrate key molecular interactions, including covalent bonds (gray), atoms (red), hydrogen bonds (dark blue), weak hydrogen bonds (light blue), and cation-π interactions (orange) (Fig. 5). Molecular docking analysis revealed that 2’’-O-galloylhyperin establishes extensive non-covalent interactions with core proteins, which are crucial for its biological activity. The specific interaction patterns are summarized below:

  • Interaction with AcsA1: A total of 14 hydrogen bonds and one cation-π interaction were formed. Specifically, the phenyl hydroxyl groups of the galloyl formed 3 hydrogen bonds with SER262 of the target protein. The galactosyl formed 6 hydrogen bonds with GLN412, LYS606, ARG512, and GLN384 (one of which with ARG512 and one with GLN384 were weak hydrogen bonds). The phenyl hydroxyl groups of the aglycone (quercetin) formed 5 hydrogen bonds with SER262, SER604, and GLY384 (one with GLY384 was a weak hydrogen bond), and a benzene ring formed 1 cation-π interaction with LYS606.

  • Interaction with PycB: A total of 17 hydrogen bonds were formed. The phenyl hydroxyl groups of the galloyl formed 4 hydrogen bonds with ALA21 and THR22 (one with ALA21 was a weak hydrogen bond). The galactosyl formed 6 hydrogen bonds with ASN304, THR339, and SER342. The phenyl hydroxyl groups of the aglycone (quercetin) formed 5 hydrogen bonds with GLN17, SER18, HIS206, and MET177 (one with SER18 was a weak hydrogen bond), along with an additional 2 weak hydrogen bonds with THR339.

  • Interaction with Pta: A total of 16 hydrogen bonds were formed. The phenyl hydroxyl groups of the galloyl formed 4 hydrogen bonds with ASN336 and GLU338 (one with ASN336 was a weak hydrogen bond). The galactosyl formed 5 hydrogen bonds with ASP46, GLY45, and LEU47 (4 of which were weak hydrogen bonds). The phenyl hydroxyl groups of the aglycone (quercetin) formed 7 hydrogen bonds with ASP226, ASP223, ARG51, PRO49, and GLY48 (3 of which, with ASP223 and GLY48, were weak hydrogen bonds).

  • Interaction with GltA: A total of 11 hydrogen bonds and one cation-π interaction were formed. The phenyl hydroxyl groups of the galloyl formed 5 hydrogen bonds with GLN411, TYR413, ILE60, TYR59, and PRO27 (those with TYR59 and PRO27 were weak hydrogen bonds). The galactosyl formed 2 hydrogen bonds with ASP63 and ARG40 (one with ARG40 was a weak hydrogen bond). The phenyl hydroxyl groups of the aglycone (quercetin) formed 4 hydrogen bonds with LYS310, ARG410, GLN232, and GLU231, and one pyran ring formed a cation-π interaction with ARG410.

  • Interaction with CfiB: A total of 10 hydrogen bonds and one cation-π interaction were formed. The phenyl hydroxyl groups of the galloyl formed 4 hydrogen bonds with GLU274, ASN234, LYS236, and GLN235 (one with GLN235 was a weak hydrogen bond), while a benzene ring formed a cation-π interaction with LYS236. The galactosyl formed 3 hydrogen bonds with ARG336. The phenyl hydroxyl groups of the aglycone (quercetin) formed 3 hydrogen bonds with SER382, GLU340, and ASN234.

Discussion

P. aeruginosa is a major pathogen responsible for bacterial pneumonia and readily develops multidrug resistance, creating severe challenges in clinical treatment [23]. Imipenem, a carbapenem antibiotic and first-line therapeutic agent for such infections, has demonstrated a marked reduction in efficacy in recent years [24, 25]. In this study, 484 medicine food homology compounds from the MCE Medicine Food Homology Compound Library (CAS: HY-CPK-15885) for their ability to reduce the MIC of imipenem against strain 18102011. A complete list was provided in Table S6. Among them, 2’’-O-galloylhyperin (1 mM) didn’t inhibit the growth of strain 18102011. A checkerboard assay was performed to determine the optimal synergistic concentrations of 2’’-O-galloylhyperin and imipenem against strain 18102011 and its transconjugant D2011. The results showed that the MIC of imipenem decreased from 4,096 µg/mL to 1,024 µg/mL in the presence of 2’’-O-galloylhyperin (8–16 µg/mL). This combination effectively inhibited the growth of strain 18102011 and D2011, demonstrating synergistic antibacterial activity (FICI ≤ 0.5). Although strain 18102011, strain D2011, and strain 2445 all carried the Class A carbapenemase KPC-2 (a serine hydrolase that utilizes serine as the catalytic nucleophile) [26], the MIC for K. pneumoniae 2445 was reduced only 2-fold (Initially, a concentration of 128 µg/mL imipenem was required to achieve a bactericidal effect (After intervention, 64 µg/mL imipenem was sufficient to significantly inhibit bacterial growth). However, 2’’-O-galloylhyperin didn’t potentiate imipenem activity against strains producing either Class B (metallo-β-lactamases, MBLs) (MBLs belong to the Ambler class B metalloenzymes and require Zn²⁺ cofactors for activity) or Class D carbapenemases (oxacillinases, OXA-type) (espite also being serine-dependent, follow distinct evolutionary trajectories and exhibit substrate specificities such as enhanced hydrolysis of oxacillin) [26, 27].

Based on these biochemical distinctions, we further evaluated the differential inhibitory effects of 2’’-O-galloylhyperin across the three carbapenemase classes (Fig. 2). A nitrocefin chromogenic assay demonstrated that 2’’-O-galloylhyperin exerted selective inhibition against KPC-2 carbapenemase, whereas its effects on MBLs and OXA-type enzymes were negligible. This enzyme-specific suppression profile aligned with the unchanged MIC values of imipenem against non-KPC-2 producers. However, the inhibitory potency of 2’’-O-galloylhyperin against KPC-2 was substantially weaker than that of the established β-lactamase inhibitor avibactam.

To elucidate the synergistic bactericidal mechanism of 2’’-O-galloylhyperin in combination with imipenem against P. aeruginosa 18102011, we conducted whole-genome transcriptome sequencing (RNA-seq) to systematically analyze transcriptional profile changes in response to imipenem treatment with or without 2’’-O-galloylhyperin. H-cluster analysis revealed that the combination significantly impaired the function of the electron transport chain (ETC) (Fig. 3). Compared to imipenem alone, the combination induced synchronized downregulation of genes encoding multiple key respiratory chain complexes, with the most severe obstruction occurring at Complex III (cytochrome bc₁ complex) and Complex IV (cytochrome C oxidase) [28–30]. Specifically, the expression of critical components (e.g., cytochrome C subunits and quinol oxidase polypeptide I) was suppressed. The combination disrupted electron transfer between Complexes III and IV, blocking electron flow and triggering negative feedback. Consequently, electron input capacity was impaired at both Complex I (NADH dehydrogenase) and Complex II (succinate-ubiquinone oxidoreductase), leading to abnormal electron accumulation within the ETC [28–31]. The resulting electron overload markedly increased ROS production [29, 30, 32, 33].

GO enrichment analysis indicated that combination significantly disrupted bacterial oxidoreductase activity and electron transport processes compared to imipenem alone (Fig. 3). Further assays demonstrated that although the T-AOC and the activities of key antioxidant enzymes, including CAT, GR, and SOD, showed no changes, intracellular ROS levels were elevated (Fig. 4). These findings suggest that 2’’-O-galloylhyperin impairs oxidoreductase function, thereby hindering the bacterial ability to produce sufficient antioxidant enzymes to counteract ROS accumulation. This could lead to membrane lipid peroxidation, altered permeability, and enhanced intracellular accumulation of imipenem, ultimately improving antibacterial efficacy [34, 35].

Iron serves as an essential cofactor for oxidoreductases (e.g., CAT) and the ETC [36], with its concentration directly modulating their activity. In groups treated with 2’’-O-galloylhyperin (alone or combined with imipenem), intracellular Fe²⁺ and Fe³⁺ levels decreased (Fig. 4). These results indicate that although Fe²⁺/Fe³⁺ redox cycling was unaffected, bacterial iron acquisition or retention was compromised. To elucidate the iron depletion mechanism, siderophore content was measured. No significant changes were observed in any treatment group, excluding impaired siderophore synthesis as the cause.

Integrated analysis of KEGG pathways and PPI networks revealed significant reprogramming of the TCA cycle and propanoate metabolism in the target bacterial strain (Fig. 5). Core proteins identified through PPI network screening included GltA, SucA, PycB, CfiB, AcsA1, and Pta, among others. Molecular docking studies demonstrated that 2’’-O-galloylhyperin exhibits high-affinity binding to GltA (citrate synthase; binding energy: -9.2 kcal·mol⁻¹), thereby upregulating gltA expression and enhancing citrate synthesis. Conversely, its interaction with PycB suppressed pycB expression (Table S5), blocking the conversion of pyruvate to oxaloacetate and creating a critical metabolic bottleneck in the TCA cycle. Notably, aconitase activity was severely impaired due to dysfunctional iron-sulfur (Fe-S) cluster cofactors (consistent with prior findings of defective iron acquisition), directly disrupting the conversion of citrate to cis-aconitate and isocitrate, leading to citrate accumulation [37–39]. Citrate accumulation directly chelates free Fe²⁺/Fe³⁺ ions, thereby exacerbating iron depletion (consistent with experimental observations) [40]. The impaired synthesis of isocitrate indirectly diminished α-ketoglutarate (α-KG) production. Although upregulated expression enhanced the conversion of α-KG to succinyl-CoA, the limited substrate supply ultimately resulted in insufficient succinyl-CoA levels. To counter this metabolic deficit, the bacterium activated a compensatory propanoate metabolism pathway: propanoate was rapidly converted to propionyl-CoA via Acs1 catalysis, followed by methyl citrate cycle-mediated generation of succinyl-CoA, temporarily alleviating the shortage of TCA cycle intermediates [41, 42]. However, the potent binding of 2’’-O-galloylhyperin to Acs1 (binding energy: -10.4 kcal·mol⁻¹) likely hyperactivated this pathway, triggering abnormal propanoate metabolic flux and compensatory overproduction of succinate. Intriguingly, despite upregulated expression of succinate dehydrogenase (SDH, Complex II), its catalytic efficiency relies on intact Fe-S clusters [43]. Under disrupted iron homeostasis, aconitase activity was severely reduced due to defects in Fe-S cluster biogenesis, which aligns with the down-regulation of its gene expression in transcriptomic data. In contrast, genes encoding succinate dehydrogenase (SDH) show up-regulated expression. This suggested that under iron restriction, limited cellular iron resources are preferentially allocated to SDH to maintain basic respiratory chain function. This selective prioritization of Fe-S cluster distribution might explain why SDH retains, albeit diminished, catalytic activity in converting succinate to fumarate.

Transcriptomic analysis revealed distinct molecular responses in the bacterial strain under combination treatment, compared to imipenem alone. Antibiotic resistance-associated gene families—including biofilm formation regulators, multidrug efflux pump systems, and β-lactamase-encoding genes—were universally up-regulated (Fig.S1). In contrast, virulence factors (e.g., exotoxin secretion-related genes) were significantly down-regulated (Fig.S1). This pattern suggested a microbial resource reallocation strategy: suppressing virulence factor synthesis to conserve energy while enhancing antibiotic tolerance mechanisms. The expression profile strongly correlated with survival stress induced by combination treatment. All highly expressed genes were enriched in the KEGG DNA damage repair pathway, consistent with elevated ROS levels. This confirmed that oxidative stress from the combination treatment triggered DNA double-strand breaks and subsequent activation of the SOS repair system [44–46] (Fig.S1). Plasmid-mediated resistance propagation exhibited divergent mechanisms (Fig.S1): The IncpRBL16 plasmid upregulated genes encoding replication initiator proteins, conjugation transfer genes, and the metallo-β-lactamase gene blaVIM−2, suggesting enhanced replicative efficiency and horizontal gene transfer potential. Conversely, the IncP6 plasmid downregulated replication-associated genes but upregulated mobilizable elements (mob gene), indicating adaptation through plasmid recombination optimization. Notably, despite upregulation of the KPC-2 carbapenemase gene (blaKPC−2), enzymatic activity decreased. This phenomenon might be due to protein misfolding.

The synergistic bactericidal mechanism of 2’’-O-galloylhyperin and imipenem involved the regulation of multiple metabolic pathways (Fig. 6). Specifically, 2’’-O-galloylhyperin inhibited the uptake of Fe²⁺/Fe³⁺ in P. aeruginosa while down-regulating pyruvate carboxylase expression, thereby impairing oxaloacetate synthesis. Notably, it also bound to citrate synthase and up-regulates its expression, accelerating the conversion of oxaloacetate to citrate. Paradoxically, despite increased citrate synthesis, restricted Fe²⁺/Fe³⁺ uptake inhibits the aconitase-catalyzed metabolism of citrate, leading to its intracellular accumulation. The accumulated citrate further chelated Fe²⁺/Fe³⁺, forming a vicious cycle that exacerbates iron depletion. Iron deficiency disrupts bacterial respiratory chain function, specifically impairing electron transfer in Complex II (succinate dehydrogenase) and Complex IV (cytochrome c oxidase), which triggers excessive ROS production. ROS accumulation induces lipid peroxidation of the cell membrane and subsequently causes conformational abnormalities in carbapenemase KPC-2. These alterations collectively compromise bacterial defense mechanisms, thereby potentiating the bactericidal effect of imipenem. This cascade of iron depletion, metabolic disruption, and oxidative stress constitutes the molecular basis of the synergy between the two compounds.

Fig. 6.

Fig. 6

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Schematic diagram illustrating the mechanism by which 2’’-O-galloylhyperin potentiates the bactericidal effect of imipenem. Note: red denotes up-regulation or activation; green denotes down- regulation or inhibition; dashed arrows indicate the direct actions or targets of 2’’-O-galloylhyperin

Conclusions and future prospects

This study first demonstrated that 2’’-O-galloylhyperin could reverse carbapenem resistance in P. aeruginosa strain 18102011. Although this compound lacked intrinsic antibacterial activity, it significantly potentiated the efficacy of imipenem. Mechanistically, 2’’-O-galloylhyperin upregulated the expression of gltA in the bacterial TCA cycle, leading to decrease intracellular levels of ferrous (Fe²⁺) and ferric (Fe³⁺) ions, downregulation of multiple iron-dependent enzyme genes, and disrupted expression of genes involved in respiratory electron transport. This cascade of events triggers excessive ROS production, thereby enhancing the bactericidal activity of imipenem. However, the cytotoxicity of 2’’-O-galloylhyperin at the tested concentrations has not been evaluated in relevant cell models, and its biosafety profile requires further investigation. In addition, although transcriptome analysis indicated that 2’’-O-galloylhyperin downregulated the expression of several virulence-associated genes in strain 18102011, these findings have not been validated by functional assays. These limitations will be addressed and further explored in future studies.

Supplementary Information

Supplementary Material 1. (5.9MB, docx)
Supplementary Material 2. (77KB, xlsx)

Acknowledgements

We are grateful to the members of the China-Japan Union Hospital, Jilin University.

Authors’ contributions

LZ: Conceptualization, Investigation, Writing– original draft, Writing– review & editing, Data curation, Validation, Visualization, Methodology. GJL: Writing– original draft, Supervision. ZXW: Writing– original draft. ZYS: Writing– original draft, Supervision, Validation. ZW: Project administration, Writing– original draft. XJG: Project administration, Writing– original draft. FYW, JL, SSZ, and YQL: Project administration, Writing– original draft. JFQ: Methodology, Investigation, Data curation, Funding acquisition, Resources, Project administration, Conceptualization, Writing– review & editing. LWZ: Methodology, Investigation, Data curation, Funding acquisition, Resources, Project administration, Conceptualization, Writing– review & editing.

Funding

The authors declare that financial support was received for this research and article publication. Funding covering study design, data collection, data analysis and publication fees was provided by the Henan Province Science and Technology Tackling Key Projects (No. 262102321126 and No. 262102310423), the Cultivation Fund for National Scientific Research Projects of Huanghuai University (XKPY-2025013), and the Qinghai Science and Technology Achievement Transformation Special Project (No. 2025-NK-112).

Data availability

The transcriptome data of strain 18102011 and its plasmids in response to imipenem treatment group vs. 2’’-O-galloylhyperin treatment group vs. combination treatment group have been uploaded to the China National Center for Bioinformatics (CNCB) (https://ngdc.cncb.ac.cn/gsub) under accession number CRA023298 (https://ngdc.cncb.ac.cn/gsa/browse/CRA023298).

Declarations

Ethics approval and consent to participate

The experimental protocols were approved by the Ethics Committee of the Jilin University (JDKQ202316EC). This study was conducted in strict accordance with the Declaration of Helsinki.

Written informed consent for publication was obtained from all participants. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lin Zheng and Ge-Jin Lu contributed equally to this work.

Contributor Information

Guang-Hui Lu, Email: 380335460@qq.com.

Jun-Feng Qi, Email: 1243377523@qq.com.

Ling-Wei Zhu, Email: lingweiz@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (5.9MB, docx)
Supplementary Material 2. (77KB, xlsx)

Data Availability Statement

The transcriptome data of strain 18102011 and its plasmids in response to imipenem treatment group vs. 2’’-O-galloylhyperin treatment group vs. combination treatment group have been uploaded to the China National Center for Bioinformatics (CNCB) (https://ngdc.cncb.ac.cn/gsub) under accession number CRA023298 (https://ngdc.cncb.ac.cn/gsa/browse/CRA023298).

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