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

2’’-O-galloylhyperin as a novel adjuvant to reduce imipenem resistance in multidrug-resistant Pseudomonas aeruginosa: Transcriptome-based mechanism of iron homeostasis disruption | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 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disruption Lin Zheng, Ge-Jin Lu, Zi-xian Wang, Zhen Wang, Zhi-Ying Shao, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8494972/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 15 You are reading this latest preprint version 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 Fe 2+ /Fe 3+ , reducing iron bioavailability, disrupting electron transfer, and increasing intracellular ROS levels in P. aeruginosa 18102011. 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. 2’’-O-galloylhyperin Pseudomonas aeruginosa imipenem oxidative damage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Pseudomonas aeruginosa is a critical pathogen in nosocomial settings, especially 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 through 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. 2 Materials and methods 2.1 Strains and Imipenem synergist screening P. aeruginosa 18102011 was isolated from the bile of a burn patient, and exhibited a resistant pattern to all 23 antibiotics tested, which could be defined as a pan-drug resistant P. aeruginosa strain [ 10 ] . Two plasmids were identified in it: the Inc pRBL16 mega-plasmid pP2011-1 carrying bla VIM−2 and the IncP6 plasmid pP2011-2 carrying bla KPC−2 [ 10 ] . A single colony was inoculated into 2 mL of Mueller-Hinton (MH) broth and incubated with shaking at 37 ℃ for 18 h. The resulting culture was diluted 1:100 in fresh MH broth and shaken until reaching the logarithmic growth phase. The bacterial suspension was then adjusted to 1 x 10 6 CFU/mL in MH broth, and 100 µL aliquots were dispensed into sterile 96-well plates. 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 1024 µg/mL) across the wells. Growth controls (bacteria without compounds) and sterility controls (media only) were included. Plates were incubated statically at 37 ℃ for 18 h, and optical density (OD) at 630 nm was measured. The fractional inhibitory concentration index (FICI) was calculated using the following formula 1.1 [ 11 ] . “ 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. FICI = \(\:\frac{\mathbf{M}\mathbf{I}\mathbf{C}\varvec{a}}{\mathbf{M}\mathbf{I}\mathbf{C}\varvec{c}}+\frac{\mathbf{M}\mathbf{I}\mathbf{C}\varvec{b}}{\mathbf{M}\mathbf{I}\mathbf{C}\varvec{d}}\) (1.1) 2.2 Strain 18102011 and D2011 growth curve under 2’’-O-galloylhyperin Single colonies of strain 18102011 and its transconjugant D2011 were separately inoculated into MH broth and cultured at 37 ℃ for 16 h. They were then sub-cultured at a 1:50 ratio in fresh MH broth and shake-incubated at 37 ℃ until the OD 600 reached 0.1. The cultures were aliquoted into sterile conical flasks and treated with different concentrations of 2’’-O-galloylhyperin (final concentrations: 0 µg/mL, 4 µg/mL, 8 µg/mL, 16 µg/mL, and 32 µg/mL), followed by continued incubation. 500 µL of culture was collected from each group every hour, and the OD value at 630 nm was recorded using a microplate reader. The entire experiment was repeated twice to ensure accuracy. The results were analyzed using Origin 2019 to plot growth curves and evaluate the effect of the 2’’-O-galloylhyperin on the growth of strain 18102011 and its transconjugant D2011. 2.3 Strain 18102011 and D2011 time-kill curve under 2’’-O-galloylhyperin and imipenem Strain 18102011 and its transconjugant D2011 were diluted with MH broth to a final concentration of 10 5 CFU/mL. Then they were evenly distributed into sterile 96-well culture plates, with each well containing 50 µL of bacterial suspension. Control groups included the following: (1) Strain without imipenem or 2’’-O-galloylhyperin; (2) Strain with 1% DMSO; (3) MH broth; (4) MH broth with imipenem at a concentration of either 1,024 µg/mL or 2,048 µg/mL; (5) MH broth with 2’’-O-galloylhyperin at a concentration of either 8 µg/mL or 16 µg/mL; (6) MH broth with 2’’-O-galloylhyperin + imipenem (e.g., 8 µg/mL + 1,024 µg/mL, 16 µg/mL + 2,048 µg/mL). At different time points (0, 1, 2, 4, 8, and 24 h), the bacterial content was determined. Details are provided below: 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. The entire experiment was repeated twice to ensure the accuracy of the results. The results were plotted as time-kill curves, with bacterial content plotted on the y-axis and time on the x-axis. 2.4 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-lactamases 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 x 10 8 CFU/mL. Cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. The cell suspension was then sonicated on ice (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 supernatant (containing the crude enzyme extract) was collected and 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. 250 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 punnett square arrangement; (4) solvent control: enzyme extract + PBS containing 1% DMSO (the highest solvent concentration used); (5) blank control: PBS + nitrocefin (to account for background absorbance); (6) 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. The entire experiment was performed in triplicate to ensure reproducibility. 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 18083286 ( bla IMP−1 ), Acinetobacter baumannii 3011 ( bla OXA ), Klebsiella oxytoca 3428 ( bla NDM−1 ), and Klebsiella pneumoniae 2445 ( bla KPC−2 ). 2.5 RNA preparation and transcriptome sequencing An overnight culture of P. aeruginosa 18102011 was diluted in fresh MH broth to final density of 1 x 10⁸ CFU/mL. Aliquots (500 µL each) of this suspension were dispensed into centrifuge tubes for the following treatment groups: (1) Negative control group: treated with an equivalent volume of MH broth; (2) solvent control group: treated with 1% 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 hours. Following incubation, cells were harvested by centrifugation at 10,000 × g for 2 min at 4°C. The supernatant was discarded, and the 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 from the cell pellets using the RNAprep Pure Cell/Bacteria Kit according to the manufacturer’s instructions. rRNA was depleted from the total RNA to enrich for mRNA. Sequencing libraries were then constructed from the enriched mRNA and subjected to paired-end sequencing on an Illumina NovaSeq platform. Three biological replicates were sequenced for each treatment group. Raw sequencing reads were quality-controlled and then mapped to the reference genomes of P. aeruginosa PA7 chromosome (GenBank: GCA_000017205.1) and its plasmids pP2011-1 (GenBank: CP116229) and pP2011-2 (GenBank: CP116230) using Bowtie2. The number of reads mapped to each gene was counted with HTSeq v0.6.1. Differential gene expression analysis was performed using the DESeq2 R package (v1.38.3). The primary comparisons were: (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 shown in formula 1.2. The resulting p-values were adjusted for multiple testing using the Benjamini-Hochberg procedure to control the false discovery rate (FDR). Genes with an adjusted p-value (FDR) 1 were considered significantly differentially expressed genes (DEGs). DEGs were visualized using volcano plots and hierarchical clustering (H-cluster) analysis. log2FoldChange= \(\:\mathbf{l}\mathbf{o}\mathbf{g}2\frac{\mathbf{t}\mathbf{r}\mathbf{e}\mathbf{a}\mathbf{t}\mathbf{m}\mathbf{e}\mathbf{n}\mathbf{t}\:\mathbf{g}\mathbf{r}\mathbf{o}\mathbf{u}\mathbf{p}}{\mathbf{c}\mathbf{o}\mathbf{n}\mathbf{t}\mathbf{r}\mathbf{o}\mathbf{l}\:\mathbf{g}\mathbf{r}\mathbf{o}\mathbf{u}\mathbf{p}}\:\) (1.2) 2.6 GO function annotation Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the Goseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. We performed GO enrichment analysis on the DEGs identified in P. aeruginosa 18102011 following 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. These graphs were built using the top five terms as central nodes, expanding to include their parent and child terms to illustrate the hierarchical relationships within the GO structure. 2.7 KEGG function annotation and key genes screening KEGG is a database resource for understanding high-level functions of the biological system, such as the cell, the organism, and ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies ( https://www.genome.jp/kegg/ ) [ 13 – 15 ] . KEGG pathway enrichment analysis of differentially expressed genes was performed using KOBAS software [ 16 ] . PPI analysis of differentially expressed genes was based on the STRING database, which contains known and predicted Protein-Protein Interactions [ 17 ] . Cytoscape software was used to analyze potential gene modules (clusters, subnetworks) within the PPI network and perform enrichment analysis to identify key genes [ 18 ] . 2.8 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 ] . The protein file in PDB format and the 3D structure of 2’’-O-galloylhyperin in SDF format were uploaded. Blind docking was selected with default settings for other parameters. CB-Dock2 automatically removed hydrogen atoms and ligands, and performed five docking runs to identify the optimal binding pocket. 2.9 Oxidoreductase, ROS and iron homeostasis assay To assess the impact of 2’’-O-galloylhyperin on the oxidative stress response and iron homeostasis of P. aeruginosa , the activities of key antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR)), intracellular ROS levels, and iron ion concentrations were measured using the following protocols. P. aeruginosa was cultured to the mid-logarithmic phase and harvested. Cells were lysed by ultrasonication on ice (200 W, cycles of 5 s on/5 s off) until complete lysis was achieved. 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 activities were calculated based on the provided formulas. All steps were performed on ice or at 4°C unless specified for incubation. 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. 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. The sample was mixed with the reagent, incubated at 37°C for 30 min in the dark, and absorbance was read at 510 nm (for Fe²⁺) and 364 nm (for Fe³⁺); (3) Siderophore Activity:​​ Assessed by 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 decrease in absorbance at 630 nm (indicating dissociation of the CAS-Fe³⁺ complex) was measured, with lower values denoting higher siderophore activity. All assays were performed in triplicate, and data are presented as mean ± standard deviation. 2.10 Nucleotide sequence accession numbers 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. 3 Results 3.1 Screening of antibiotic synergists This study screened 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. The complete list of compounds is provided in Table S1. More than 25% of the compounds demonstrated antibacterial activity against strain 18102011 when imipenem was absent (Table S1). Among these, 2’’-O-galloylhyperin (1 mM) did not inhibit the growth of strain 18102011. However, in combination with a sub-inhibitory concentration of imipenem, it showed potential synergistic effects, as indicated by reduced turbidity in the culture medium. Therefore, a punnett square was performed to determine the optimal synergistic concentrations of 2’’-O-galloylhyperin and imipenem against strain 18102011. The assay revealed that the MIC of imipenem decreased from 4,096 µg/mL to 1,024 µg/mL in the presence of 2’’-O-galloylhyperin (8 or 16 µg/mL). This combination effectively inhibited the growth of strain 18102011 and its transconjugant E. coli D2011, demonstrating synergistic antibacterial activity (FICI ≤ 0.5). These results indicate that 2’’-O-galloylhyperin can effectively restore imipenem’s antibacterial activity against these resistant strains. 2’’-O-galloylhyperin did not enhance the antibacterial activity of imipenem against the following resistant strains, as evidenced by unchanged MIC values: P. aeruginosa 18083286 carrying class B carbapenemase IMP-1 and class D carbapenemase OXA-50; A. baumannii 3011 carrying class D carbapenemase; K. oxytoca carrying class B carbapenemase NDM-1. However, at a concentration of 128 μg/mL, 2’’-O-galloylhyperin reduced the imipenem MIC value by 2-fold in K. pneumoniae 2445 carrying class A carbapenemase KPC-2, suggesting a potential selective synergistic effect against specific carbapenemase types (Table S2). 3.2 Growth profile 2’’-O-galloylhyperin didn’t significantly inhibit the growth of P. aeruginosa 18102011 or its transconjugant E. coli 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 hours. 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. In comparison, the transconjugant E. coli D2011 exhibited a slower growth rate, entering the logarithmic phase at 6 h and reaching the plateau phase at 7 h. The inhibitory activity of 2’’-O-galloylhyperin against strains 18102011 and D2011 showed no significant concentration dependence over the range of 4-32 µg/mL. 3.3 time-kill curve The growth profile indicated that 2’’-O-galloylhyperin alone didn’t inhibit the growth of P. aeruginosa 18102011 or E. coli D2011 (Fig.1). To further investigate this, the study evaluated the synergistic bactericidal effect of 2’’-O-galloylhyperin and imipenem using time-kill assays (Fig.1). Neither 2’’-O-galloylhyperin (8 μg/mL) nor imipenem (1,024 μg/mL) alone exhibited inhibitory effects against the tested strains ( P. aeruginosa 18102011 and E. coli D2011) (Fig.1). However, the combination of 2’’-O-galloylhyperin (8 μg/mL) with imipenem (1,024 μg/mL) produced a significant inhibitory effect that persisted for at least 8 h. The results indicate that while 2’’-O-galloylhyperin itself does not suppress the growth of strain 18102011 or D2011, it effectively restores the bactericidal activity of imipenem against these strains. 3.4 The effect of 2’’-O-galloylhyperin on β-lactamase activity This study evaluated the inhibitory effects of 2’’-O-galloylhyperin and avibactam on various carbapenemases using nitrocefin-based assays, monitored by changes in optical density (OD) (Fig.2). The results were strain-specific and revealed diverse interaction patterns between the two compounds: In the E. coli D2011 transconjugant (co-harboring bla KPC-2 and bla VIM-2 ), both compounds inhibited KPC-2 and/or VIM-2 activity, as indicated by significantly lower OD values compared to the untreated control. 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 bla IMP-1 ), neither compound inhibited IMP-1 activity, increased OD values confirmed this result; In Acinetobacter baumannii 3011 (carrying bla OXA ), both compounds individually strongly inhibited OXA activity. However, their combination unexpectedly eliminated the inhibitory effect, indicating a potential antagonistic interaction; In Klebsiella oxytoca 3428 (carrying bla NDM-1 ), neither compound significantly inhibited NDM-1 activity; In Klebsiella pneumoniae 2445 (carrying bla KPC-2 ), both 2’’-O-galloylhyperin and avibactam significantly inhibited KPC-2 activity. However, their combination failed to show synergy. 3.5 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 18102011 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. 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-4S 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 CARB 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-relatedgenes, 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. 3.6 Oxidoreductase-electron transfer activity GO enrichment analysis GO enrichment analysis revealed that the combination induced significant gene expression changes in P. aeruginosa 18102011compared 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 18102011. 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. 3.7 propionate 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 . Compared to the strain 18102011 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. 4. Discussion P. aeruginosa ​​is a major pathogen responsible for bacterial pneumonia and readily develops multidrug resistance, posing significant 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, we screened 484 medicine food homology compounds and identified 2’’-O-galloylhyperin as a potent synergist that reduces the MIC of imipenem by 4-fold against strain 18102011 and its carbapenem-resistant transconjugants. Despite lacking direct antibacterial activity, it effectively functioned as an antibiotic adjuvant. However, 2’’-O-galloylhyperin did not enhance the efficacy of imipenem against strains harboring other types of carbapenemases; notably, the MIC for carrying KPC-2 was reduced only 2-fold. Strain 18102011, its transconjugant D2011, and strain 2445 carried the Class A carbapenemase KPC-2, a serine hydrolase that utilizes serine as the catalytic nucleophile [ 26 ] . In contrast, other tested strains exclusively produced either Class B (metallo-β-lactamases, MBLs) or Class D carbapenemases (oxacillinases, OXA-type). MBLs belong to the Ambler class B metalloenzymes and require Zn²⁺ cofactors for activity, whereas OXA-type enzymes, despite 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 reactive oxygen species (ROS) production [ 29 , 30 , 32 , 33 ] . 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. 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 electron transport chain [ 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 propionate 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 propionate metabolism pathway: propionate 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 propionate 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 suggests 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 may 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 suggests 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 confirms 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 VIM-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 ( bla KPC−2 ), enzymatic activity decreased. This phenomenon may be due to protein misfolding. The synergistic bactericidal mechanism of 2’’-O-galloylhyperin and imipenem involves the regulation of multiple metabolic pathways. Specifically, 2’’-O-galloylhyperin inhibits the uptake of Fe²⁺/Fe³⁺ in P. aeruginosa while down-regulating pyruvate carboxylase expression, thereby impairing oxaloacetate synthesis. Notably, it also binds 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 chelates 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 KPC-2 carbapenemase. 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.​ 5. Conclusions and future prospects This study evaluated the potential of 2’’-O-galloylhyperin to reverse carbapenem resistance in P. aeruginosa strain 18102011. The results demonstrated that although the compound itself lacked intrinsic antibacterial activity, it significantly enhanced the efficacy of imipenem in combination treatment. Notably, 2’’-O-galloylhyperin also effectively downregulated the expression of virulence-associated genes in this strain. Mechanistically, 2’’-O-galloylhyperin upregulated GltA in the bacterial TCA cycle, leading to reduced levels of ferrous (Fe²⁺) and ferric (Fe³⁺) ions, downregulation of multiple iron-dependent enzyme genes, and disruption of respiratory chain electron transport-related gene expression. This integrated cascade of events triggers excessive ROS production, thereby potentiating the bactericidal effect of imipenem. 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. Consent for Publication Not Applicable Funding The author declare financial support was received for the research and/or publication of this article. Funding for the study design, data collection, data generation, and publication costs was provided by Qinghai Science and Technology Achievement Transformation Special Project (No. 2025-NK-112) and the National Science and Natural Science Foundation of China (Grant agreement: 31872486). Author Contribution 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. 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08:13:31","extension":"html","order_by":35,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":175306,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/a3961019be16aa6900eaa685.html"},{"id":100265831,"identity":"3233ff91-cc16-4e27-b541-3b31510bf499","added_by":"auto","created_at":"2026-01-14 18:16:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32707520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth and time-kill curve of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e 18102011 and transconjugant D2011\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enote: (a, b) Growth curves of \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 (a) and \u003cem\u003eE. coli\u003c/em\u003e D2011 (b). The horizontal axis represents the incubation time (h), and the vertical axis represents the bacterial optical density (OD\u003csub\u003e600\u003c/sub\u003e). Lines represent the following treatments: DMSO control (32 μg/mL, red), and 2’’-O-galloylhyperin at 0 μg/mL (black), 4 μg/mL (blue), 8 μg/mL (green), 16 μg/mL (purple), and 32 μg/mL (yellow).\u003c/p\u003e\n\u003cp\u003e(c, d) Time-kill curves of \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 (c) and \u003cem\u003eE. coli\u003c/em\u003e D2011 (d). The horizontal axis represents the incubation time (h), and the vertical axis represents the bacterial count (CFU/mL). Lines represent the same treatments as in panels a and b.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/0f7f748e342a489d35b38116.png"},{"id":100372483,"identity":"105c14ae-33fe-44a5-bc60-43621b2fcf66","added_by":"auto","created_at":"2026-01-16 08:12:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":554029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of 2’’-O-galloylhyperin and avibactam on enzyme activity in different types of bacteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enote: a. \u003cem\u003eE. coli\u003c/em\u003e D2011 (including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e); b. \u003cem\u003eP. aeruginosa\u003c/em\u003e 18083286 (including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-1\u003c/sub\u003e); c. \u003cem\u003eA. baumannii\u003c/em\u003e 3011 (including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e); d. \u003cem\u003eK. oxytoca\u003c/em\u003e 3428 (including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e); e. \u003cem\u003eK. pneumoniae\u003c/em\u003e 2455 (including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e). A: bacterial lysis supernatant; B: bacterial lysis supernatant + avibactam (50 μg/mL); C: bacterial lysis supernatant + avibactam (50 μg/mL) + 2’’-O-galloylhyperin (8 μg/mL); D: bacterial lysis supernatant + 2’’-O-galloylhyperin (8 μg/mL). One-way ANOVA indicated that differences between treatment groups were statistically significant (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/419bef93600b6fc049eefa0f.png"},{"id":100371096,"identity":"b3593adc-d48c-4dbe-bb37-e728b7971fd6","added_by":"auto","created_at":"2026-01-16 08:09:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1603603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional enrichment analysis of DEGs between the combination treatment group and the imipenem alone group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enote: a. volcano plot illustrating transcriptional differences between the combination treatment group and the imipenemalone group; b. Heatmap displaying gene expression patterns across all samples, where the horizontal axis represents sample names a1-a3: \u003cem\u003eP. aeruginosa \u003c/em\u003e18102011 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. Gene Ontology (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 \u0026lt; 0.05); d. A Directed Acyclic Graph (DAG) illustrating the hierarchical relationships of enriched GO terms, where the top five significant GO terms (p \u0026lt; 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)).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/5cd516dcec26d98de96f319b.png"},{"id":100372513,"identity":"b1e2705f-ac07-4ded-8ce2-f9f2fa84cc94","added_by":"auto","created_at":"2026-01-16 08:12:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15597553,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eViolin plots depicting the results of oxidoreductase activity, ROS levels, and iron homeostasis-related assays in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. aeruginosa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e strain 18102011 under different treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enote: a. T-AOC; b. SOD activity; c. CAT activity; d. GR activity; e. intracellular ROS levels; f. siderophore production assessed by CAS assay; g. Fe\u003csup\u003e2+\u003c/sup\u003e levels; h. Fe\u003csup\u003e3+\u003c/sup\u003e levels; i. Ferric reducing antioxidant power. A: control (no drug); B: DMSO (8 μg/mL); C: 2’’-O-galloylhyperin (8 μg/mL); D: imipenem (512 μg/mL); E: Combination treament (imipenem, 512 μg/mL + 2’’-O-galloylhyperin, 8 μg/mL).\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/419cd46996abd8c195b9de93.png"},{"id":100265822,"identity":"5b37be71-4169-436d-8d59-435bb8a8338b","added_by":"auto","created_at":"2026-01-14 18:16:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1647224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular docking of 2’’-O-galloylhyperin with key proteins in propionate metabolism and TCA cycle KEGG pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enote: 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 18082011’s propionate metabolism and TCA cycle; c. the molecular docking between core genes (propionate metabolism/TCA cycle) and 2’’-O-galloylhyperin: (1) \u003cem\u003egltA\u003c/em\u003e and 2’’-O-galloylhyperin; (2) \u003cem\u003epycB\u003c/em\u003e and 2’’-O-galloylhyperin; (3) \u003cem\u003esucA\u003c/em\u003e and 2’’-O-galloylhyperin; (4)\u003cem\u003e acsA1\u003c/em\u003e and 2’’-O-galloylhyperin; (5) \u003cem\u003ecfiB\u003c/em\u003e and 2’’-O-galloylhyperin; (6) \u003cem\u003epta\u003c/em\u003e and 2’’-O-galloylhyperin.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/d8d593f6dd8f8b1f6e3e378b.png"},{"id":100371929,"identity":"a69a9876-bedd-4d07-9533-8b9f770d8cda","added_by":"auto","created_at":"2026-01-16 08:11:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1670117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram illustrating the mechanism by which 2’’-O-galloylhyperin potentiates the bactericidal effect of imipenem\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enote: red denotes up-regulation or activation; green denotes down- regulation or inhibition; dashed arrows indicate the direct actions or targets of 2’’-O-galloylhyperin.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/f635e001630c202ca24708ec.png"},{"id":100265826,"identity":"a7d0ebb3-fa31-4f3e-9bd7-b788baf13ff1","added_by":"auto","created_at":"2026-01-14 18:16:31","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":76172,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1new.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8494972/v1/87cb6845edd49279d7fb7f99.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"2’’-O-galloylhyperin as a novel adjuvant to reduce imipenem resistance in multidrug-resistant Pseudomonas aeruginosa: Transcriptome-based mechanism of iron homeostasis disruption","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e is a critical pathogen in nosocomial settings, especially ICUs, where it causes severe, often fatal infections due to its biofilm formation and rapid acquisition of antibiotic resistance \u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. The overuse of broad-spectrum antibiotics has led to the emergence of strains resistant to last-line carbapenems, primarily through carbapenemase production \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eIn the search for novel therapeutic strategies, natural compounds have garnered significant interest. 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin, a primary bioactive flavonoid from \u003cem\u003ePyrola\u003c/em\u003e species, possesses significant antioxidant and anti-inflammatory properties due to its galloyl-pyrogallol structure \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Its structural analog, quercetin, is known to inhibit biofilm formation in pathogens like \u003cem\u003eStreptococcus mutans\u003c/em\u003e, \u003cem\u003eP. aeruginosa\u003c/em\u003e, and \u003cem\u003eSalmonella Typhimurium\u003c/em\u003e \u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Despite this potential, the ability of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin to reverse or reduce carbapenem resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e has not been investigated. Therefore, this study aims to evaluate the efficacy of this compound in enhancing imipenem against \u003cem\u003eP. aeruginosa\u003c/em\u003e and to elucidate its underlying molecular mechanisms.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Strains and Imipenem synergist screening\u003c/h2\u003e \u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 was isolated from the bile of a burn patient, and exhibited a resistant pattern to all 23 antibiotics tested, which could be defined as a pan-drug resistant \u003cem\u003eP. aeruginosa\u003c/em\u003e strain \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Two plasmids were identified in it: the Inc\u003csub\u003epRBL16\u003c/sub\u003e mega-plasmid pP2011-1 carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM\u0026minus;2\u003c/sub\u003e and the IncP6 plasmid pP2011-2 carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. A single colony was inoculated into 2 mL of Mueller-Hinton (MH) broth and incubated with shaking at 37 ℃ for 18 h. The resulting culture was diluted 1:100 in fresh MH broth and shaken until reaching the logarithmic growth phase. The bacterial suspension was then adjusted to 1 x 10\u003csup\u003e6\u003c/sup\u003e CFU/mL in MH broth, and 100 \u0026micro;L aliquots were dispensed into sterile 96-well plates. 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 1024 \u0026micro;g/mL) across the wells.\u003c/p\u003e \u003cp\u003eGrowth controls (bacteria without compounds) and sterility controls (media only) were included. Plates were incubated statically at 37 ℃ for 18 h, and optical density (OD) at 630 nm was measured. The fractional inhibitory concentration index (FICI) was calculated using the following formula 1.1 \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. \u0026ldquo;\u003cem\u003ea\u003c/em\u003e\u0026rdquo; represents the MIC of imipenem in combination; \u0026ldquo;\u003cem\u003eb\u003c/em\u003e\u0026rdquo; represents the MIC of the compound in combination; \u0026ldquo;\u003cem\u003ec\u003c/em\u003e\u0026rdquo; represents the MIC of the imipenem alone; and \u0026ldquo;\u003cem\u003ed\u003c/em\u003e\u0026rdquo; represents the MIC of the compound alone. Synergy was defined as FICI\u0026thinsp;\u0026le;\u0026thinsp;0.5, no interaction as 0.5\u0026thinsp;\u0026lt;\u0026thinsp;FICI\u0026thinsp;\u0026lt;\u0026thinsp;4, and antagonism as FICI\u0026thinsp;\u0026gt;\u0026thinsp;4.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFICI =\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\mathbf{M}\\mathbf{I}\\mathbf{C}\\varvec{a}}{\\mathbf{M}\\mathbf{I}\\mathbf{C}\\varvec{c}}+\\frac{\\mathbf{M}\\mathbf{I}\\mathbf{C}\\varvec{b}}{\\mathbf{M}\\mathbf{I}\\mathbf{C}\\varvec{d}}\\)\u003c/span\u003e\u003c/span\u003e \u003cb\u003e(1.1)\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Strain 18102011 and D2011 growth curve under 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin\u003c/h2\u003e \u003cp\u003eSingle colonies of strain 18102011 and its transconjugant D2011 were separately inoculated into MH broth and cultured at 37 ℃ for 16 h. They were then sub-cultured at a 1:50 ratio in fresh MH broth and shake-incubated at 37 ℃ until the OD\u003csub\u003e600\u003c/sub\u003e reached 0.1. The cultures were aliquoted into sterile conical flasks and treated with different concentrations of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin (final concentrations: 0 \u0026micro;g/mL, 4 \u0026micro;g/mL, 8 \u0026micro;g/mL, 16 \u0026micro;g/mL, and 32 \u0026micro;g/mL), followed by continued incubation. 500 \u0026micro;L of culture was collected from each group every hour, and the OD value at 630 nm was recorded using a microplate reader. The entire experiment was repeated twice to ensure accuracy. The results were analyzed using Origin 2019 to plot growth curves and evaluate the effect of the 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin on the growth of strain 18102011 and its transconjugant D2011.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Strain 18102011 and D2011 time-kill curve under 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin and imipenem\u003c/h2\u003e \u003cp\u003eStrain 18102011 and its transconjugant D2011 were diluted with MH broth to a final concentration of 10\u003csup\u003e5\u003c/sup\u003e CFU/mL. Then they were evenly distributed into sterile 96-well culture plates, with each well containing 50 \u0026micro;L of bacterial suspension. Control groups included the following: (1) Strain without imipenem or 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin; (2) Strain with 1% DMSO; (3) MH broth; (4) MH broth with imipenem at a concentration of either 1,024 \u0026micro;g/mL or 2,048 \u0026micro;g/mL; (5) MH broth with 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin at a concentration of either 8 \u0026micro;g/mL or 16 \u0026micro;g/mL; (6) MH broth with 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin\u0026thinsp;+\u0026thinsp;imipenem (e.g., 8 \u0026micro;g/mL\u0026thinsp;+\u0026thinsp;1,024 \u0026micro;g/mL, 16 \u0026micro;g/mL\u0026thinsp;+\u0026thinsp;2,048 \u0026micro;g/mL). At different time points (0, 1, 2, 4, 8, and 24 h), the bacterial content was determined. Details are provided below:\u003c/p\u003e \u003cp\u003eSamples from different groups were collected and serially diluted in MH broth to 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e through 10\u003csup\u003e\u0026minus;\u0026thinsp;9\u003c/sup\u003e. 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. The entire experiment was repeated twice to ensure the accuracy of the results. The results were plotted as time-kill curves, with bacterial content plotted on the y-axis and time on the x-axis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Nitrocefin hydrolysis\u003c/h2\u003e \u003cp\u003eNitrocefin 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-lactamases activity by quantitatively monitoring this colorimetric shift \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Details are provided below:\u003c/p\u003e \u003cp\u003eStrains 18102011 and D2011 were cultured in MH broth and adjusted to final concentration of 1 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL. Cells were harvested by centrifugation, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. The cell suspension was then sonicated on ice (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 \u003cem\u003eg\u003c/em\u003e for 2 min at 4 ℃, and the supernatant (containing the crude enzyme extract) was collected and 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 \u0026micro;L of the crude enzyme extract and 135 \u0026micro;L of PBS containing the appropriate drugs to achieve the desired final concentrations. The following experimental groups were established:\u003c/p\u003e \u003cp\u003e(1) negative control (100% enzyme activity): enzyme extract\u0026thinsp;+\u0026thinsp;PBS (no inhibitor); (2) positive control (inhibition control): enzyme extract\u0026thinsp;+\u0026thinsp;avibactam (final conc. 250 \u0026micro;g/mL); (3) test groups: enzyme extract\u0026thinsp;+\u0026thinsp;imipenem (final conc. 0, 1, 2, 4, 8, 16, 32, 64, 256, and 1024 \u0026micro;g/mL); enzyme extract\u0026thinsp;+\u0026thinsp;2\u0026rsquo;\u0026rsquo;-O-galloylhyperin (final conc. 0, 2, 4, 8, 32, 64, and 128 \u0026micro;g/mL); enzyme extract\u0026thinsp;+\u0026thinsp;imipenem\u0026thinsp;+\u0026thinsp;2\u0026rsquo;\u0026rsquo;-O-galloylhyperin in a punnett square arrangement; (4) solvent control: enzyme extract\u0026thinsp;+\u0026thinsp;PBS containing 1% DMSO (the highest solvent concentration used); (5) blank control: PBS\u0026thinsp;+\u0026thinsp;nitrocefin (to account for background absorbance); (6) substrate control: MH broth\u0026thinsp;+\u0026thinsp;nitrocefin (optional, to check for non-enzymatic hydrolysis).\u003c/p\u003e \u003cp\u003eThe plate was pre-incubated at 37 ℃ for 3 min. The reaction was initiated by adding 75 \u0026micro;L of nitrocefin solution (final concentration 75 \u0026micro;g/mL) to each well, bringing the total reaction volume to 200 \u0026micro;L. 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. The entire experiment was performed in triplicate to ensure reproducibility.\u003c/p\u003e \u003cp\u003eTo further assess the inhibitory spectrum of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin, the assay was repeated using a panel of clinically relevant carbapenemase-producing strains: \u003cem\u003eP. aeruginosa\u003c/em\u003e 18083286 (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP\u0026minus;1\u003c/sub\u003e), \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e 3011 (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e), \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e 3428 (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e), and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e 2445 (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 RNA preparation and transcriptome sequencing\u003c/h2\u003e \u003cp\u003eAn overnight culture of \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 was diluted in fresh MH broth to final density of 1 x 10⁸ CFU/mL. Aliquots (500 \u0026micro;L each) of this suspension were dispensed into centrifuge tubes for the following treatment groups:\u003c/p\u003e \u003cp\u003e(1) Negative control group: treated with an equivalent volume of MH broth; (2) solvent control group: treated with 1% DMSO; (3) imipenem treatment group: treated with imipenem at a final concentration of 512 \u0026micro;g/mL; (4) 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin treatment group: treated with 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin at a final concentration of 8 \u0026micro;g/mL; (5) combination treatment group: treated with imipenem (512 \u0026micro;g/mL)\u0026thinsp;+\u0026thinsp;2\u0026rsquo;\u0026rsquo;-O-galloylhyperin (8 \u0026micro;g/mL).\u003c/p\u003e \u003cp\u003eAll tubes were incubated statically at 37\u0026deg;C for 7 hours. Following incubation, cells were harvested by centrifugation at 10,000 \u0026times; g for 2 min at 4\u0026deg;C. The supernatant was discarded, and the cell pellets were resuspended in 100 \u0026micro;L 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 from the cell pellets using the RNAprep Pure Cell/Bacteria Kit according to the manufacturer\u0026rsquo;s instructions. rRNA was depleted from the total RNA to enrich for mRNA. Sequencing libraries were then constructed from the enriched mRNA and subjected to paired-end sequencing on an Illumina NovaSeq platform. Three biological replicates were sequenced for each treatment group.\u003c/p\u003e \u003cp\u003eRaw sequencing reads were quality-controlled and then mapped to the reference genomes of \u003cem\u003eP. aeruginosa\u003c/em\u003e PA7 chromosome (GenBank: GCA_000017205.1) and its plasmids pP2011-1 (GenBank: CP116229) and pP2011-2 (GenBank: CP116230) using Bowtie2. The number of reads mapped to each gene was counted with HTSeq v0.6.1. Differential gene expression analysis was performed using the DESeq2 R package (v1.38.3). The primary comparisons were: (1) Imipenem treatment group vs. negative control group; (2) 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin treatment group vs. negative control group; and (3) Combination treatment group \u003cem\u003evs\u003c/em\u003e. negative control group. For each gene, the log2 fold change (log2FoldChange) in expression was calculated as shown in formula 1.2. The resulting p-values were adjusted for multiple testing using the Benjamini-Hochberg procedure to control the false discovery rate (FDR). Genes with an adjusted p-value (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and |log2FoldChange| \u0026gt; 1 were considered significantly differentially expressed genes (DEGs). DEGs were visualized using volcano plots and hierarchical clustering (H-cluster) analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003elog2FoldChange=\u003c/b\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\mathbf{l}\\mathbf{o}\\mathbf{g}2\\frac{\\mathbf{t}\\mathbf{r}\\mathbf{e}\\mathbf{a}\\mathbf{t}\\mathbf{m}\\mathbf{e}\\mathbf{n}\\mathbf{t}\\:\\mathbf{g}\\mathbf{r}\\mathbf{o}\\mathbf{u}\\mathbf{p}}{\\mathbf{c}\\mathbf{o}\\mathbf{n}\\mathbf{t}\\mathbf{r}\\mathbf{o}\\mathbf{l}\\:\\mathbf{g}\\mathbf{r}\\mathbf{o}\\mathbf{u}\\mathbf{p}}\\:\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003e(1.2)\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 GO function annotation\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the Goseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. We performed GO enrichment analysis on the DEGs identified in \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 following combination treatment (2\u0026rsquo;\u0026rsquo;-O-galloylhyperin\u0026thinsp;+\u0026thinsp;imipenem) \u003cem\u003evs\u003c/em\u003e 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. These graphs were built using the top five terms as central nodes, expanding to include their parent and child terms to illustrate the hierarchical relationships within the GO structure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 KEGG function annotation and key genes screening\u003c/h2\u003e \u003cp\u003eKEGG is a database resource for understanding high-level functions of the biological system, such as the cell, the organism, and ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e[\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. KEGG pathway enrichment analysis of differentially expressed genes was performed using KOBAS software \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePPI analysis of differentially expressed genes was based on the STRING database, which contains known and predicted Protein-Protein Interactions \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Cytoscape software was used to analyze potential gene modules (clusters, subnetworks) within the PPI network and perform enrichment analysis to identify key genes \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Molecular docking of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin with key proteins in significantly enriched KEGG pathways\u003c/h2\u003e \u003cp\u003eThe crystal structures of key proteins from the significantly enriched KEGG pathways of \u003cem\u003eP. aeruginosa\u003c/em\u003e were obtained from the RCSB.PDB database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The ligand structure file of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin was retrieved from the PubChem database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Molecular docking between the protein and 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin was performed using CB-Dock2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cadd.labshare.cn/cb-dock2/php/index.php\u003c/span\u003e\u003cspan address=\"https://cadd.labshare.cn/cb-dock2/php/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. The protein file in PDB format and the 3D structure of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin in SDF format were uploaded. Blind docking was selected with default settings for other parameters. CB-Dock2 automatically removed hydrogen atoms and ligands, and performed five docking runs to identify the optimal binding pocket.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Oxidoreductase, ROS and iron homeostasis assay\u003c/h2\u003e \u003cp\u003eTo assess the impact of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin on the oxidative stress response and iron homeostasis of \u003cem\u003eP. aeruginosa\u003c/em\u003e, the activities of key antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR)), intracellular ROS levels, and iron ion concentrations were measured using the following protocols.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e was cultured to the mid-logarithmic phase and harvested. Cells were lysed by ultrasonication on ice (200 W, cycles of 5 s on/5 s off) until complete lysis was achieved. The lysate was centrifuged at 10,000 x g for 15 min at 4\u0026deg;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\u0026rsquo;s protocols. Absorbance was measured at the wavelength specified for each respective kit, and activities were calculated based on the provided formulas. All steps were performed on ice or at 4\u0026deg;C unless specified for incubation.\u003c/p\u003e \u003cp\u003eIntracellular ROS levels were quantified using a Reactive Oxygen Species Assay Kit. Bacteria treated with 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin for 6 h were collected, washed with PBS, and incubated with 10 \u0026micro;mol/L DCFH-DA at 37\u0026deg;C for 20 min in the dark. Cells were then washed thoroughly with PBS to remove excess probe. Fluorescence was measured at Excitation/Emission\u0026thinsp;=\u0026thinsp;488/522 nm. ROS levels were expressed relative to the untreated control.\u003c/p\u003e \u003cp\u003eIron 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\u0026deg;C for 20 min, centrifuged, and the absorbance of the supernatant was measured at 700 nm; (2) Fe\u0026sup2;⁺/Fe\u0026sup3;⁺ Ratio:​​ Determined by the 1,10-phenanthroline method. The sample was mixed with the reagent, incubated at 37\u0026deg;C for 30 min in the dark, and absorbance was read at 510 nm (for Fe\u0026sup2;⁺) and 364 nm (for Fe\u0026sup3;⁺); (3) Siderophore Activity:​​ Assessed by 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 decrease in absorbance at 630 nm (indicating dissociation of the CAS-Fe\u0026sup3;⁺ complex) was measured, with lower values denoting higher siderophore activity. All assays were performed in triplicate, and data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Nucleotide sequence accession numbers\u003c/h2\u003e \u003cp\u003eThe transcriptome data of strain 18102011 and its plasmids in response to imipenem treatment group \u003cem\u003evs\u003c/em\u003e. 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin treatment group \u003cem\u003evs\u003c/em\u003e. combination treatment group have been uploaded to the China National Center for Bioinformatics (CNCB) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/gsub\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/gsub\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) under accession number CRA023298.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Screening of antibiotic synergists\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study screened 484 medicine food homology compounds\u0026nbsp;from the MCE\u0026nbsp;Medicine Food Homology Compound Library (CAS: HY-CPK-15885)\u0026nbsp;for their ability to reduce the MIC of imipenem against strain 18102011.\u0026nbsp;The complete list of compounds is provided in Table S1.\u003c/p\u003e\n\u003cp\u003eMore than 25% of the compounds demonstrated antibacterial activity against strain 18102011 when imipenem was absent (Table S1). Among these,\u0026nbsp;2’’-O-galloylhyperin (1 mM) did not inhibit the growth of strain 18102011. However, in combination with a sub-inhibitory concentration of imipenem, it showed potential synergistic effects, as indicated by reduced turbidity in the culture medium. Therefore, a punnett square was performed to determine the optimal synergistic concentrations of 2’’-O-galloylhyperin and imipenem against strain 18102011. The assay revealed that the MIC of imipenem decreased from 4,096 µg/mL to 1,024 µg/mL in the presence of 2’’-O-galloylhyperin (8 or 16 µg/mL). This combination effectively inhibited the growth of strain 18102011 and its transconjugant \u003cem\u003eE. coli\u003c/em\u003e D2011, demonstrating synergistic antibacterial activity (FICI ≤ 0.5). These results indicate that 2’’-O-galloylhyperin can effectively restore imipenem’s antibacterial activity against these resistant strains.\u003c/p\u003e\n\u003cp\u003e2’’-O-galloylhyperin did not enhance the antibacterial activity of imipenem against the following resistant strains, as evidenced by unchanged MIC values: \u003cem\u003eP. aeruginosa\u003c/em\u003e 18083286 carrying class B carbapenemase IMP-1 and class D carbapenemase OXA-50; \u003cem\u003eA. baumannii\u003c/em\u003e 3011 carrying class D carbapenemase; \u003cem\u003eK. oxytoca\u003c/em\u003e carrying class B carbapenemase NDM-1. However, at a concentration of 128 μg/mL, 2’’-O-galloylhyperin reduced the imipenem MIC value by 2-fold in \u003cem\u003eK. pneumoniae\u003c/em\u003e 2445 carrying class A carbapenemase KPC-2, suggesting a potential selective synergistic effect against specific carbapenemase types (Table S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Growth profile\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2’’-O-galloylhyperin didn’t significantly inhibit the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 or its transconjugant \u003cem\u003eE. coli\u003c/em\u003e 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 hours. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn comparison, the transconjugant \u003cem\u003eE. coli\u003c/em\u003e D2011 exhibited a slower growth rate, entering the logarithmic phase at 6 h and reaching the plateau phase at 7 h. The inhibitory activity of\u0026nbsp;2’’-O-galloylhyperin against strains 18102011 and D2011 showed no significant concentration dependence over the range of 4-32 µg/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etime-kill curve\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth profile indicated that 2’’-O-galloylhyperin alone didn’t inhibit the growth of \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 or \u003cem\u003eE. coli\u003c/em\u003e D2011 (Fig.1). To further investigate this, the study evaluated the synergistic bactericidal effect of 2’’-O-galloylhyperin and imipenem using time-kill assays (Fig.1). Neither 2’’-O-galloylhyperin (8 μg/mL) nor imipenem (1,024 μg/mL) alone exhibited inhibitory effects against the tested strains (\u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011 and \u003cem\u003eE. coli\u003c/em\u003e D2011) (Fig.1). However, the combination of 2’’-O-galloylhyperin (8 μg/mL) with imipenem (1,024 μg/mL) produced a significant inhibitory effect that persisted for at least 8 h.\u003c/p\u003e\n\u003cp\u003eThe results indicate that while 2’’-O-galloylhyperin itself does not suppress the growth of strain 18102011 or D2011, it effectively restores the bactericidal activity of imipenem against these strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 The effect of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e2’’-O-galloylhyperin on β-lactamase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study evaluated the inhibitory effects of 2’’-O-galloylhyperin and avibactam on various carbapenemases using nitrocefin-based assays, monitored by changes in optical density (OD) (Fig.2). The results were strain-specific and revealed diverse interaction patterns between the two compounds:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eD2011 transconjugant (co-harboring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eVIM-2\u003c/sub\u003e), both compounds inhibited KPC-2 and/or VIM-2 activity, as indicated by significantly lower OD values compared to the untreated control. Although avibactam demonstrated superior efficacy, its combination with 2’’-O-galloylhyperin resulted in weaker inhibition than avibactam alone, suggesting potential interference; In \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003e18083286 (carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eIMP-1\u003c/sub\u003e), neither compound inhibited IMP-1 activity, increased OD values confirmed this result; In \u003cem\u003eAcinetobacter baumannii\u0026nbsp;\u003c/em\u003e3011 (carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eOXA\u003c/sub\u003e), both compounds individually strongly inhibited OXA activity. However, their combination unexpectedly eliminated the inhibitory effect, indicating a potential antagonistic interaction; In \u003cem\u003eKlebsiella oxytoca\u0026nbsp;\u003c/em\u003e3428 (carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e), neither compound significantly inhibited NDM-1 activity; In \u003cem\u003eKlebsiella pneumoniae\u0026nbsp;\u003c/em\u003e2445 (carrying \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC-2\u003c/sub\u003e), both 2’’-O-galloylhyperin and avibactam significantly inhibited KPC-2 activity. However, their combination failed to show synergy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Effect of 2’’-O-galloylhyperin on the transcriptional level of strain 18102011\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter 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.\u003c/p\u003e\n\u003cp\u003eFigure 3 presents volcano plots illustrating DEG distributions in strain 18102011 under imipenem alone and combination. The x-axis represents the log2 fold-change (combination \u003cem\u003evs\u003c/em\u003e. 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.\u003c/p\u003e\n\u003cp\u003eH-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-4S iron-sulfur cluster domain) (Table S4). These findings suggest the antibacterial effect may involve disruption of the electron transport chain via transcriptional suppression.\u003c/p\u003e\n\u003cp\u003eWe compared the expression of virulence factors (based on the VFDB database) and resistance genes (based on the CARB database) in \u003cem\u003eP. aeruginosa\u003c/em\u003e 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 \u003cem\u003eP. aeruginosa\u0026nbsp;\u003c/em\u003evirulence-relatedgenes, 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Oxidoreductase-electron transfer activity GO enrichment analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGO enrichment analysis revealed that the combination induced significant gene expression changes in \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011compared 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.\u003c/p\u003e\n\u003cp\u003eA 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 \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7\u003c/strong\u003e \u003cstrong\u003epropionate metabolism and TCA cycle KEGG pathways\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo 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 \u003cem\u003egltA\u003c/em\u003e, 2-oxoglutarate dehydrogenase E1 component gene \u003cem\u003esucA\u003c/em\u003e, pyruvate carboxylase subunit B gene \u003cem\u003epycB\u003c/em\u003e, and 2-oxoglutarate carboxylase small subunit gene \u003cem\u003ecfiB\u003c/em\u003e; for the propanoate metabolism pathway, they were acetyl-coenzyme A synthetase 1 gene \u003cem\u003eacsA1\u003c/em\u003e, phosphate acetyltransferase gene \u003cem\u003epta\u003c/em\u003e, and pyruvate dehydrogenase E1 component subunit alpha gene \u003cem\u003epdhA\u0026nbsp;\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eCompared to the strain 18102011 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\u003cem\u003e\u0026nbsp;gltA\u003c/em\u003e and \u003cem\u003esucA\u003c/em\u003e were significantly up-regulated, whereas those of \u003cem\u003epycB\u003c/em\u003e and \u003cem\u003ecfiB\u003c/em\u003e were significantly down-regulated. In the propanoate metabolism pathway, the expression of \u003cem\u003eacsA1\u003c/em\u003e was significantly up-regulated, while the expression of \u003cem\u003epta\u003c/em\u003e and \u003cem\u003epdhA\u003c/em\u003e was significantly down-regulated (Table S5).\u003c/p\u003e\n\u003cp\u003eTo 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:\u003c/p\u003e\n\u003cp\u003eInteraction with AcsA1:\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eInteraction with PycB:\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eInteraction with Pta:\u0026nbsp;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).\u003c/p\u003e\n\u003cp\u003eInteraction with GltA:\u0026nbsp;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.\u003c/p\u003e\n\u003cp\u003eInteraction 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.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eP. aeruginosa\u003c/em\u003e ​​is a major pathogen responsible for bacterial pneumonia and readily develops multidrug resistance, posing significant challenges in clinical treatment \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Imipenem, a carbapenem antibiotic and first-line therapeutic agent for such infections, has demonstrated a marked reduction in efficacy in recent years \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In this study, we screened 484 medicine food homology compounds and identified 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin as a potent synergist that reduces the MIC of imipenem by 4-fold against strain 18102011 and its carbapenem-resistant transconjugants. Despite lacking direct antibacterial activity, it effectively functioned as an antibiotic adjuvant. However, 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin did not enhance the efficacy of imipenem against strains harboring other types of carbapenemases; notably, the MIC for carrying KPC-2 was reduced only 2-fold.\u003c/p\u003e \u003cp\u003eStrain 18102011, its transconjugant D2011, and strain 2445 carried the Class A carbapenemase KPC-2, a serine hydrolase that utilizes serine as the catalytic nucleophile \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. In contrast, other tested strains exclusively produced either Class B (metallo-β-lactamases, MBLs) or Class D carbapenemases (oxacillinases, OXA-type). MBLs belong to the Ambler class B metalloenzymes and require Zn\u0026sup2;⁺ cofactors for activity, whereas OXA-type enzymes, despite also being serine-dependent, follow distinct evolutionary trajectories and exhibit substrate specificities such as enhanced hydrolysis of oxacillin \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on these biochemical distinctions, we further evaluated the differential inhibitory effects of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin across the three carbapenemase classes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A nitrocefin chromogenic assay demonstrated that 2\u0026rsquo;\u0026rsquo;-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\u0026rsquo;\u0026rsquo;-O-galloylhyperin against KPC-2 was substantially weaker than that of the established β-lactamase inhibitor avibactam.\u003c/p\u003e \u003cp\u003eTo elucidate the synergistic bactericidal mechanism of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin in combination with imipenem against \u003cem\u003eP. aeruginosa\u003c/em\u003e18102011, we conducted whole-genome transcriptome sequencing (RNA-seq) to systematically analyze transcriptional profile changes in response to imipenem treatment with or without 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin. H-cluster analysis revealed that the combination significantly impaired the function of the electron transport chain (ETC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). 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) \u003csup\u003e[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. 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 \u003csup\u003e[\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The resulting electron overload markedly increased reactive oxygen species (ROS) production \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eGO enrichment analysis indicated that combination significantly disrupted bacterial oxidoreductase activity and electron transport processes compared to imipenem alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings suggest that 2\u0026rsquo;\u0026rsquo;-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 \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIron serves as an essential cofactor for oxidoreductases (e.g., CAT) and the electron transport chain \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, with its concentration directly modulating their activity. In groups treated with 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin (alone or combined with imipenem), intracellular Fe\u0026sup2;⁺ and Fe\u0026sup3;⁺ levels decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicate that although Fe\u0026sup2;⁺/Fe\u0026sup3;⁺ 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.\u003c/p\u003e \u003cp\u003eIntegrated analysis of KEGG pathways and PPI networks revealed significant reprogramming of the TCA cycle and propionate metabolism in the target bacterial strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Core proteins identified through PPI network screening included GltA, SucA, PycB, CfiB, AcsA1, and Pta, among others. Molecular docking studies demonstrated that 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin exhibits high-affinity binding to GltA (citrate synthase; binding energy: -9.2 kcal\u0026middot;mol⁻\u0026sup1;), 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 \u003csup\u003e[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Citrate accumulation directly chelates free Fe\u0026sup2;⁺/Fe\u0026sup3;⁺ ions, thereby exacerbating iron depletion (consistent with experimental observations) \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. 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 propionate metabolism pathway: propionate 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 \u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. However, the potent binding of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin to Acs1 (binding energy: -10.4 kcal\u0026middot;mol⁻\u0026sup1;) likely hyperactivated this pathway, triggering abnormal propionate 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 \u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. 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 suggests 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 may explain why SDH retains, albeit diminished, catalytic activity in converting succinate to fumarate.\u003c/p\u003e \u003cp\u003eTranscriptomic analysis revealed distinct molecular responses in the bacterial strain under combination treatment, compared to imipenem alone. Antibiotic resistance-associated gene families\u0026mdash;including biofilm formation regulators, multidrug efflux pump systems, and β-lactamase-encoding genes\u0026mdash;were universally up-regulated (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In contrast, virulence factors (e.g., exotoxin secretion-related genes) were significantly down-regulated (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This pattern suggests 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 confirms that oxidative stress from the combination treatment triggered DNA double-strand breaks and subsequent activation of the SOS repair system \u003csup\u003e[\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Plasmid-mediated resistance propagation exhibited divergent mechanisms (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e): The IncpRBL16 plasmid upregulated genes encoding replication initiator proteins, conjugation transfer genes, and the metallo-β-lactamase gene VIM-2, suggesting enhanced replicative efficiency and horizontal gene transfer potential. Conversely, the IncP6 plasmid downregulated replication-associated genes but upregulated mobilizable elements (\u003cem\u003emob\u003c/em\u003e gene), indicating adaptation through plasmid recombination optimization. Notably, despite upregulation of the KPC-2 carbapenemase gene (\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e), enzymatic activity decreased. This phenomenon may be due to protein misfolding.\u003c/p\u003e \u003cp\u003eThe synergistic bactericidal mechanism of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin and imipenem involves the regulation of multiple metabolic pathways. Specifically, 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin inhibits the uptake of Fe\u0026sup2;⁺/Fe\u0026sup3;⁺ in \u003cem\u003eP. aeruginosa\u003c/em\u003e while down-regulating pyruvate carboxylase expression, thereby impairing oxaloacetate synthesis. Notably, it also binds to citrate synthase and up-regulates its expression, accelerating the conversion of oxaloacetate to citrate. Paradoxically, despite increased citrate synthesis, restricted Fe\u0026sup2;⁺/Fe\u0026sup3;⁺ uptake inhibits the aconitase-catalyzed metabolism of citrate, leading to its intracellular accumulation. The accumulated citrate further chelates Fe\u0026sup2;⁺/Fe\u0026sup3;⁺, 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 KPC-2 carbapenemase. 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.​\u003c/p\u003e "},{"header":"5. Conclusions and future prospects","content":"\u003cp\u003eThis study evaluated the potential of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin to reverse carbapenem resistance in \u003cem\u003eP. aeruginosa\u003c/em\u003e strain 18102011. The results demonstrated that although the compound itself lacked intrinsic antibacterial activity, it significantly enhanced the efficacy of imipenem in combination treatment. Notably, 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin also effectively downregulated the expression of virulence-associated genes in this strain. Mechanistically, 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin upregulated GltA in the bacterial TCA cycle, leading to reduced levels of ferrous (Fe\u0026sup2;⁺) and ferric (Fe\u0026sup3;⁺) ions, downregulation of multiple iron-dependent enzyme genes, and disruption of respiratory chain electron transport-related gene expression. This integrated cascade of events triggers excessive ROS production, thereby potentiating the bactericidal effect of imipenem.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent for Publication\u003c/h2\u003e \u003cp\u003eNot Applicable\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThe author declare financial support was received for the research and/or publication of this article. Funding for the study design, data collection, data generation, and publication costs was provided by Qinghai Science and Technology Achievement Transformation Special Project (No. 2025-NK-112) and the National Science and Natural Science Foundation of China (Grant agreement: 31872486).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLZ: Conceptualization, Investigation, Writing\u0026ndash; original draft, Writing\u0026ndash; review \u0026amp; editing, Data curation, Validation, Visualization, Methodology. GJL: Writing\u0026ndash; original draft, Supervision. ZXW: Writing\u0026ndash; original draft. ZYS: Writing\u0026ndash; original draft, Supervision, Validation. ZW: Project administration, Writing\u0026ndash; original draft. XJG: Project administration, Writing\u0026ndash; original draft. FYW, JL, SSZ, and YQL: Project administration, Writing\u0026ndash; original draft. JFQ: Methodology, Investigation, Data curation, Funding acquisition, Resources, Project administration, Conceptualization, Writing\u0026ndash; review \u0026amp; editing. LWZ: Methodology, Investigation, Data curation, Funding acquisition, Resources, Project administration, Conceptualization, Writing\u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe are grateful to the members of the China-Japan Union Hospital, Jilin University.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe transcriptome data of strain 18102011 and its plasmids in response to imipenem treatment group vs. 2\u0026rsquo;\u0026rsquo;-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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBlanc DS, Petignat C, Janin B, Bille J, Francioli P. 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Genetics. 2015;201(4):1349\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1534/genetics.115.178970\u003c/span\u003e\u003cspan address=\"10.1534/genetics.115.178970\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"2’’-O-galloylhyperin, Pseudomonas aeruginosa, imipenem, oxidative damage","lastPublishedDoi":"10.21203/rs.3.rs-8494972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8494972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImipenem, a critical antibiotic for treating multidrug-resistant \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, now faces severe resistance from this pathogen. This study investigates the synergistic effects and underlying mechanisms of the combination of 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin with imipenem against \u003cem\u003eP. aeruginosa.\u003c/em\u003e 2\u0026rsquo;\u0026rsquo;-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\u0026thinsp;\u0026le;\u0026thinsp;0.5, synergy). This compound upregulates the citrate synthase gene \u003cem\u003egltA\u003c/em\u003e, and downregulates the aconitase gene \u003cem\u003eacnA\u003c/em\u003e, enhancing citric acid synthesis while inhibiting its dehydration to cis-aconitase acid. Citric acid chelates Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e, reducing iron bioavailability, disrupting electron transfer, and increasing intracellular ROS levels in \u003cem\u003eP. aeruginosa\u003c/em\u003e 18102011. Additionally, 2\u0026rsquo;\u0026rsquo;-O-galloylhyperin reduces the activity of the carbapenemase KPC-2. These findings highlight its potential as an adjuvant to enhance imipenem efficacy against \u003cem\u003eP. aeruginosa\u003c/em\u003e infections.\u003c/p\u003e","manuscriptTitle":"2’’-O-galloylhyperin as a novel adjuvant to reduce imipenem resistance in multidrug-resistant Pseudomonas aeruginosa: Transcriptome-based mechanism of iron homeostasis disruption","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 18:16:26","doi":"10.21203/rs.3.rs-8494972/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-14T04:23:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T09:31:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35092124314382822342426788644548443443","date":"2026-04-04T04:33:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81609490033261308268269763003649747798","date":"2026-04-04T03:18:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28123439169534831861403867109333158353","date":"2026-04-03T15:35:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322811461244501452744419974838401793794","date":"2026-04-03T09:35:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31044203553093660580115293572067929983","date":"2026-04-02T03:39:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171714038775627388712243059848628003544","date":"2026-04-02T03:24:59+00:00","index":"hide","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-01T10:55:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-23T13:18:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"89383142560716170174437168774910086945","date":"2026-01-14T14:03:44+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-12T18:02:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-06T07:34:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-05T12:02:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2026-01-05T11:43:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26d304c4-da0c-495b-a9a5-3937febaa8eb","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-08T01:53:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 18:16:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8494972","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8494972","identity":"rs-8494972","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}