Introduction

Bloodstream infections (BSIs) represent a significant global public health concern, characterized by a high mortality rates1. In Europe, an estimated 1,200,000 BSI episodes occur annually, leading to long-term sequelae2. BSIs are often caused by catheter devices, including Hemodialysis catheterand central venous catheters3,4. These catheter-related infections are frequently caused by skin commensals, with gram-positive organisms accounting for 70% of cases4. Among these, Staphylococcus epidermidis (S. epidermidis) is the predominant pathogen associated with both implanted device infections and BSIs5. Alarmingly, S. epidermidis infections frequently exhibit antibiotic resistance, with multiple resistance in nosocomial strains reported in 70–85% of cases6,7. Furthermore, a large proportion of S. epidermidis isolates are methicillin-resistant (methicillin-resistant Staphylococcus Epidermidis; MRSE), and demonstrating extensive-resistance to β-lactam antibiotics7. Vancomycin is a restricted selection of available treatment options8. However, it has limited biocidal efficacy, necessitates therapeutic drug monitoring, and may induce nephrotoxicity and/or ototoxicity in specific patient populations9,10. Therefore, the current prevalence of MRSE-associated BSIs presents a significant therapeutic challenge, emphasizing the need for research on more effective and safer therapeutic agents11.

“Traditional Chinese medicines (TCM)”, derived from plants and historically used across diverse cultures, are commonly safe when administered in appropriate dosages12. Consequently, there is a renewed focus on exploring traditional or herbal medicines as potential alternatives or complementary treatments for emerging and re-emerging multidrug-resistant bacteria13. Numerous traditional and herbal medicines have been studied for their antimicrobial activity14,15,16,17. Aqueous extracts of Carum copticum and Albizia adianthifolia exhibited notable antibacterial effects against multidrug-resistant gram-negative human pathogenic bacteria, including Escherichia coli, Pseudomonas species, Klebsiella pneumoniae Salmonella species, and Proteus species18,19. Similarly, in our previous study, Patrinia scabiosaefolia exhibited strong antibacterial effects against MRSE both in vitro and in vivo20. Therefore, we speculate that other traditional or herbal medicines may also have effective antibacterial activity against MRSE-associated BSIs.

Emilia sonchifolia (L.) DC(E. sonchifolia) has a well-documented history of use in China, as recorded in the "Lingnan Medicine Collection." It is primarily used in TCM for its anti-inflammatory, analgesic, antibacterial, antiviral, antitumor, hypoglycemic, and immune-regulating properties21. Notably, E. sonchifolia has demonstrated significant antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Neisseria gonorrhoeae in vitro21. However, its antibacterial activity and mechanisms of action against MRSE remain unknown.

Contemporary research increasingly employs multi-omics approaches to study microorganisms22,23,24. Proteomics is employed in antibacterial research to elucidate antimicrobial mechanisms, screen potential drugs, and facilitate vaccine development23. Similarly, transcriptomics is applied to explore antibacterial mechanisms, identify therapeutic targets, and support the development of novel drugs by analyzing of gene expression profiles25. The application of proteomics and transcriptomics has remarkably advanced our understanding of bacterial mechanisms26. Hence, integrating these techniques in antibacterial research enables a comprehensive understanding of changes in bacterial protein expression and gene regulation. This approach aids in elucidating antibacterial mechanisms and identifying novel antibacterial targets.

Therefore, this systematic study in vitro and in vivo antibacterial activities of E. sonchifolia against MRSE. Additionally, transcriptomic and proteomic approaches were employed to elucidate the potential mechanisms underlying its antibacterial effects on MRSE.

Materials and methods

Materials

Methicillin-resistant Staphylococcus Epidermidis(MRSE) strains were cultured and maintained in our laboratory. The ethanol extract of E. sonchifolia (10 g crude drug/1 g extract) was purchased from Shanxi Xibolan Biotechnology Co., Ltd. (20,220,315). Assay kits for AMP, GMP, and IMP were acquired from Beijing APPLYGEN Gene Technology Co., Ltd.. Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) were obtained from Beijing Solarbio Science & Technology Co., Ltd.. Forty male C57 mice were used: C57BL/6 J mice (n = 40, both from Changsha Tianqin Biotechnology Co., Ltd.) were used. At the time of the test the mice were 6–8 weeks old and had body weights of 20.17 ± 1.21 g, respectively (mean ± SEM).

Methods

Culture conditions

The MRSE strains were stored at a temperature of -80℃ in Tryptic Soy Broth (TSB) supplemented with 20% (vol/vol) glycerol. Before each experiment, approximately 20 µL aliquots of the frozen cultures were transferred to tubes containing 5 mL of TSB and incubated overnight at 37℃. Subsequently, the cultures were inoculated onto Tryptic Soy Agar (TSA) and stored at 4℃ until they were needed for further experimentation.

The antibacterial activity of E. Sonchifolia in vitro

Determination of minimum inhibitory concentration (MIC)

The MIC of E. sonchifolia extract was determined using the broth dilution method according to the Clinical and Laboratory Standards Institute of the United States of America (CLSI). In brief, a bacterial suspension cultured to the logarithmic phase was diluted to a concentration of 1 × 106 colony-forming units per milliliter (CFU/mL). Then, 100 µL of the adjusted inoculum (1 × 106 CFU/mL) was added to each well of a 96-well Microtiter plate. Subsequently, 100 µL of serial twofold dilutions of E. sonchifolia with TSB were dispensed in the wells, ranging from a concentration of 80 mg/mL to 0.0195 mg/mL. After incubation for 18 h at 37℃, the MIC was defined as the lowest concentration of luteo.

Determination of minimum bactericidal concentration (MBC)

The broth dilution method was used to determine the MBC of E. sonchifolia27. The culture suspensions (100 L) of MRSE treated with E. sonchifolia at concentrations of 5 and 40 mg/mL, where no bacterial growth was observed, were evenly spread on TSA plates and incubated at 37℃ for 18 h. Surviving colonies were subsequently observed. The MBC was defined as the lowest concentration of an antimicrobial agent capable of inactivating > 99.99% of the bacterial population (< 10 CFU/mL). The assay was performed in triplicates.

The antibacterial activity of E. Sonchifolia in vivo

Animals conditioning

The mice were assigned to individual cages in a pathogen-free facility with a maximum occupancy of six mice per cage. Prior to their utilization, the mice were subjected to a 12-h light–dark cycle at a temperature of 22 ± 2 °C for a duration of one week. They were provided unrestricted access to food and water.

Establishment of mouse immunosuppressive models

Based on the existing literature, a mouse immunosuppressive model was established by intraperitoneal injection of Cyclophosphamide solution at 120 mg/kg body weight for 4 days. Control mice were administered phosphate-buffered saline(PBS). Subsequently, the thymus and spleen were extracted from both immunosuppressed and control mice, and the thymus and spleen indices were computed.

Treatment of E. Sonchifolia against MRSE-induced BSIs

The animals were randomly assigned to different groups prior to the experiment, including group A(Immunosuppressive, no BSI, no treatment), group B(Immunosuppressive, BSI, no treatment), group C (Immunosuppressive, BSI, E. sonchifolia), and group D(Non- Immunosuppressive, no-BSI, no treatment). In the group C, E. sonchifolia was administered orally at a dose of 8 g/kg 7 days prior to infection. The other three groups received an equal volume of PBS.

The immunosuppression model was implemented three days prior to infection, following the aforementioned method, in the group A, group B, and group C, the group D was treated with PBS. Furthermore, the group B and group C were infected 7 days after E. sonchifolia oral administration by tail vein injection with 1 × 108 cfu/mL MRSE, and the group A and group D were treated with PBS. The tail vein method was consistent with that described by Liu et al.28. At 12 h post infection (pi), blood samples were collected from the tail vein of all groups. The mice were euthanized with 100 mg/kg pentobarbitol sodium (IP). Subsequently, the aforementioned samples underwent serial dilution in PBS and were subsequently plated on TSA agar plates for a duration of 24 h at a temperature of 37 °C. Ultimately, the enumeration of clones was conducted to assess the efficacy of the mouse BSI models29.

The aforementioned mice experiments were approved by the University Committee on Use and Care of Animals at the Guizhou University of Traditional Chinese Medicine in accordance with the Laboratory Animal Guidelines for Ethical Review of Animal Welfare (GB/T 35892—2018). All experiments were performed in accordance with relevant guidelines and regulations.

The study is reported in accordance with ARRIVE guidelines.

Proteomics study

TMT-based quantitative proteomic analyses were performed as described20.

Proteins were extracted from the treated (using E. sonchifolia) and untreated cultures of MRSE with 1/2 MIC (2.5 mg/mL) for 4 h and were prepared which were sent to LC-BIO Technologies Co. Ltd. (Hangzhou, Zhejiang, China) for proteomic analyses. Protein identification, quantification, classification, and interaction prediction were performed as previously described23. The raw files generated by AQ Exactive Plus were converted using Proteome Discoverer 2.1 (Thermo Fisher Scientific) and sent to OmicStudio tools (Lc-bio Technologies Co., Ltd.) for analysis30. Differentially expressed proteins were defined as those with a fold change (FC) ≥ 1.2 or ≤ 1/1.2 -fold and a P-value < 0.05. Differentially expressed proteins were functionally classified using Gene Ontology (GO) terms (http://www.omicsbean.com). We used the KEGG database (www.kegg.jp/feedback/copyright.html) for the pathway enrichment analysis31.

Transcriptomics study

Transcriptome sequencing was performed as Cheng described32 . Proteins were extracted from the treated (using E. sonchifolia) and untreated cultures of MRSE with 1/2 MIC (2.5 mg/mL) for 4 h and were prepared which were sent to LC-BIO Technologies Co. Ltd. (Hangzhou, Zhejiang, China) for transcriptome sequencing.

After estimating the expression levels of all transcripts and analyzing the expression levels for mRNAs, the differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 with p < 0.05, by DESeq2, GO, and KEGG enrichment of the differentially activated pathways were analyzed. We used the KEGG database (www.kegg.jp/feedback/copyright.html) for the pathway enrichment analysis31.

Transcriptomic and proteomic integrative analysis methods

Transcriptomic and proteomic analyses were performed to identify the expression of genes and potential antibacterial mechanisms of E. sonchifolia against MRSE.

Effect of E. sonchifolia on purine metabolism

The activities of IMP, GPM, and AMP were evaluated in MRSE under E. sonchifolia stress. MRSE in the logarithmic growth phase was introduced to E. sonchifolia at a MIC. The mixture was then incubated at 37 °C for 4 h. A negative control consisting of PBS was prepared. The mixtures were then subjected to centrifugation at 12,000 revolutions per minute for 5 min at 4 °C, and the resulting supernatant was discarded. The cells were then washed and resuspended in PBS. Subsequently, the samples were subjected to ultrasonication at a concentration of 30% for a duration of 30 min (with an interval of 5 s) and then centrifuged at a speed of 12,000 revolutions per minute for 5 min at a temperature of 4℃. Finally, the supernatant was divided into aliquots and used for IMP, GPM, and AMP assays using kits from Beijing APPLYGEN Gene Technology Co., Ltd.

Effect of E. sonchifolia on antioxidant system

The activities of superoxide dismutase (SOD), catalase (CAT) were evaluated in MRSE under E. sonchifolia stress. MRSE in the logarithmic growth phase was introduced to E. sonchifolia at a MIC. The mixture was then incubated at 37 °C for 4 h. A negative control consisting of PBS was prepared. The mixtures were then subjected to centrifugation at 12,000 revolutions per minute for 5 min at 4 °C, and the resulting supernatant was discarded. The cells were then washed and resuspended in PBS. Subsequently, the samples were subjected to ultrasonication at a concentration of 30% for a duration of 30 min (with an interval of 5 s) and then centrifuged at a speed of 12,000 revolutions per minute for 5 min at a temperature of 4℃. The supernatant was divided into aliquots and used for CAT and SOD assays using kits from Beijing Solarbio Science and Technology Co.,Ltd.

Effect of E. sonchifolia on morphological characterizations of MRSE

Morphological characterization of MRSE treated with E. sonchifolia was performed using Scanning Electron Microscopy (SEM) Analysis Transmission Electron Microscopy (TEM) Analysis. SEM was performed using a modified version of a previously reported method. In summary, E. sonchifolia (1 × MIC) was introduced into a logarithmic phase bacterial suspension. The control group received an equal volume of PBS. All groups were incubated at 37℃ for a duration of 4 h. Subsequently, the treated bacterial cells were harvested, washed, and subsequently fixed in a 3% glutaraldehyde solution overnight at 4℃. Next, the bacterial cells were dehydrated by applying varying concentrations of ethanol, followed by replacement with tertiary butyl alcohol. Subsequently, the samples were freeze-dried, sputter-coated with gold, and examined using a scanning electron microscope. For TEM, the MRSE was treated in a manner consistent with the SEM procedure. As per a previous report, the specimens were affixed to a fibrous carbon film and observed using TEM33.

Statistical analysis

Values are reported as means ± standard deviation (SDs). One-way analysis of variance (ANOVA) was conducted to assess statistical differences among various groups. Significant differences between means were determined using Tukey’s Honest Significant Difference test, with the significance level set at p < 0.05.

Results

The antibacterial activity of E. Sonchifolia in vitro

The antibacterial activities of E. sonchifolia against MRSE were primarily indicated by MIC and MBC. After 18 h of incubation at 37℃, turbidity was noticed in the well 2.5 to 0.039 mg/ml of E. sonchifolia. Turbidity was absent at concentrations ranging from 40 to 5 mg/ml, the absence of turbidity was observed, indicating an inhibitory effect on bacterial proliferation. According to CLSI, the MIC of E. sonchifolia against MRSE was 5 mg/mL (Table 1).

Table 1 MIC and MBC value of E. sonchifolia against MRSE isolates.
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MRSE was resistant to E. sonchifolia, confirming its efficacy. Suspensions ranging from 40 to 5 mg/mL were cultured onto TSA agar plates and subjected to an 18 h incubation period. An observation of less than 10 CFU/mL in the concentration range of 40–20 mg/mL served as conclusive evidence of its bactericidal properties. Therefore, the MBC value of E. sonchifolia was 20 mg/mL (Table 1).

The antibacterial activity of E. sonchifolia in vivo

E. sonchifolia exhibited superior in vitro antibacterial activity against MRSE, as demonstrated in previous studies. To further assess its antibacterial efficacy in vivo, E. Sonchifolia was administered to treat MRSE-induced bloodstream infections (Fig. 1a). An immunosuppression model was used prior to the establishment of MRSE-induced BSIs. Spleen and thymus indices were used to evaluate the immunosuppression model. Figures 1b and c demonstrate a significant reduction in the spleen and thymus indices of the group A, group B, and group C when compared to the group D , that demonstrated that the model was successfully established. Based on the immunosuppression model, BSIs were induced by MRSE. Additionally, the quantification of bacteria in the blood served as a standard measure to assess the antibacterial efficacy of E. Sonchifolia. As shown in Fig. 1d, a notable decrease in the number of clones in the blood was observed in group C compared to that of the group B, indicating the significantly superior in vivo antibacterial activity of E. sonchifolia.

Fig. 1
figure 1

Investigation of the antibacterial activity of Emilia sonchifolia in vivo. (a) Experimental scheme determining the MRSE-induced BSIs in mice treated with E. sonchifolia. (b) Determination of spleen index in the group A, group B, group C, and group D. (c) Determination of the thymus index in the group A, group B, group C, and group D. (d) Bacterial load in blood 24 h post-infection. The x-axis indicates the mice treated with or without E. sonchifolia. Data are expressed as mean (± SD) of eight replicates (compared with the control, * p < 0.05, ** p < 0.01).

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Proteomic analysis of MRSE following E. sonchifolia treatment

The effect of E. sonchifolia extract on MRSE at the protein level was evaluated using a TMT-based quantitative method. A total of 1684 proteins were identified in MRSE in this study. Differentially expressed proteins (DEPs) were defined as follows: fold-change (FC) > 1.20 or < 0.80 and p < 0.05. The comparison of control and E sonchifolia-treated MRSE revealed 113 significant DEPs (64 upregulated proteins and 49 downregulated proteins), volcano plots of FC values against p-values (two-tailed Student’s t-test), and the number of differentially expressed proteins and genes (Fig. 2a–d). GO functional enrichment analysis of the DEPs revealed that the 15 most significantly enriched GO terms were mainly BPs and MFs, including the oxidation–reduction process (GO:0055114),integral component of the membrane (GO:0016021), and cytoplasm (GO:0005737) (p < 0.001; Fig. 2e. KEGG functional enrichment analysis of the DEPs showed that the most significantly enriched metabolic pathways were biosynthesis of secondary metabolites, biosynthesis of antibiotics, microbial metabolism in diverse environments, and carbon metabolism.

Fig. 2
figure 2

Significantly differential proteins of MRSE in 5 mg/mL E. sonchifolia stress using TMT-based quantitative proteomics. (a) The number of DEPs red represent up-regulated proteins, and green represent down-regulated proteins. (b) The horizontal axis is the relative quantitative protein value after Log2 logarithm conversion, and the vertical axis is the difference significance test p-value value after -Log10 logarithm conversion. The red dots indicate up-regulated proteins, and blue dots indicate down-regulated proteins. (c) Hierarchical cluster analysis of differentially expressed proteins between MRSE from control and treated with E. sonchifolia. (d) The number of proteins in different subcellular localization in the proteomics between MRSE from control and treated with E. sonchifolia. (e,f) Go annotation and KEGG pathway of DEPs. (g) Enrichment plot of connectivity between differentially expressed down-regulated proteins on the left and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the right. Each line represents overlap between pairwise comparisons, based on gene set enrichment analysis. (h) KEGG pathway analysis of the up-regulated of KEGG.

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Biosynthesis of amino acids, purine metabolism is shown in Fig. 2f, Moreover, KEGG pathway analysis revealed components of 21 KEGG pathways among the 33 downregulated proteins, the majority of which were associated with urine metabolism, cationic antimicrobial peptide (CAMP) resistance, two-component system (TCS), and metabolic pathways (Fig. 2g), and upregulated KEGG pathways were associated with ribosome and nitrogen metabolism (Fig. 2h).

Transcriptomic analysis of MRSE upon E. sonchifolia treatment

The effects of E. sonchifolia treatment on the MRSE transcriptome were determined by RNA-seq experiments. A total of 2353 mRNA were identified in this study Differentially expressed genes (DEGs) were defined as follows: corrected p-value < 0.05 and |log2 FC|≥ 1. Comparison of the control and E. sonchifolia -treated MRSE revealed 511 DEGs (299 upregulated and 212 downregulated genes) (Fig. 3a–c).

Fig. 3
figure 3figure 3

Remarkably differential proteins of MRSE in 5 mg/mL E. sonchifolia stress using Transcriptomic. (a) Number of DEPs: red represents up-regulated proteins, and green represents down-regulated proteins. (b) Horizontal axis depicts the relative quantitative protein value after Log2 logarithm conversion, and the vertical axis presents the difference significance test p-value value after -Log10 logarithm conversion. The red dots indicate up-regulated proteins, and blue dots indicate down-regulated proteins. (c) Hierarchical cluster analysis of differentially expressed proteins between MRSE from control and those treated with E. sonchifolia. (d,e) GO annotation and KEGG pathway of DEPs. (f) Energy metabolism down-regulates energy metabolism-related pathways in mRNA. (g) KEGG pathway analysis of the down-regulated.

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GO annotation analysis revealed 1421 functional terms, including 308 MFs, 28 CCs, and 197 BPs. GO functional enrichment analysis of the DEGs showed that most of the top-enriched terms were BPs and MFs, including phosphorylation (GO:0016310), integral components of the membrane (GO:0016021), cytoplasm (GO:0005737), and metal ion binding (GO:0046872) (Fig. 3d). KEGG pathway annotation and enrichment analysis indicated that the DEGs were enriched in several pathways including energy metabolism, amino acid metabolism, biosynthesis of other secondary metabolites, carbohydrate metabolism, and metabolism of cofactors and vitamins (Fig. 3e).

Moreover, KEGG pathway analysis revealed components of the KEGG pathways among the 212 downregulated proteins, the majority of which were associated with metabolism, including arginine biosynthesis, thiamine metabolism, glyoxylate and dicarboxylate metabolism, lysine biosynthesis (Fig. 3f); and environmental Information Processing (ABC transporters) (Fig. 3g).

Transcriptomic and proteomic integrative analysis

Targeted transcriptomic data based on proteomic pathways were related to pathways such as mapko00230 (purine metabolism), mapko02020 (TCS) , and mapko01100 (metabolic pathways; Fig. 4).

Fig. 4
figure 4

Targeted transcriptomic data based on proteomic pathways were found to be related to pathways with string network of down-regulated proteins.

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The antibacterial mechanism of E. sonchifolia in vitro

Effect of E. sonchifolia on purine metabolism

The purine metabolism pathway plays an important role in bacterial survival and adaptation to the environment. Recently, researchers have discovered a number of new enzymes and metabolic pathways involved in bacterial purine metabolism such as GMP, AMP and IMP played important role in purine metabolism. In this study, we found that the activities of GMP, AMP, IMP decreased after exposure of MIC E. sonchifolia for 4 h (Figs. 5a–c). These findings indicate that E. sonchifolia can inhibit GMP, AMP, and IMP activities in MRSE.

Fig. 5
figure 5

Effect of E. sonchifolia on purine metabolism of MRSE. (a) Effect of E. sonchifolia on GMP concentration in MRSE. (b) Effect of E. sonchifolia on AMP concentration in MRSE. (c) Effect of E. sonchifolia on IMP concentration in MRSE. Data are presented as the mean (± SD) of three replicates (compared with the control, *p < 0.01, **p < 0.01).

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Effect of E. sonchifolia on antioxidant system

Antioxidant levels can alleviate harmful reactive oxygen species (ROS)34. In contrast, high antioxidant levels are considered to be the mechanism of action of antibacterial agents. Levels of antioxidant molecules, such as CAT and SOD, play important roles in the antioxidant system. In this study, we found that the activities of CAT and SOD decreased by 73% and 90%, respectively, after exposure of MIC E. sonchifolia for 4 h (Fig. 6a and b). These findings indicate that E. sonchifolia inhibits SOD and CAT activity in MRSE.

Fig. 6
figure 6

Effect of Emilia sonchifolia on defense mechanisms of MRSE. (a) Effect of E. sonchifolia on CAT activity in MRSE. (b) Effect of E. sonchifolia on SOD activity in MRSE.

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SEM analysis

To examine the alterations in the cellular morphology of MRSE following treatment with E. sonchifolia, we conducted MRSE observations SEM. In the control group (Fig. 7a), MRSE cells presented a regular shape, smooth surface, and an intact surface. After subjecting the bacteria to an incubation period of 4 h with 5 mg/mL E. sonchifolia, notable alterations in the morphology of the bacterial cells were observed, as depicted in Fig. 7b. The surface of the MRSE with E. sonchifolia exhibited irregular and rugged characteristics, with certain areas exhibiting extensive fractures that resulted in the release of the contents.

Fig. 7
figure 7

Scanning electron microcopy observations of morphology changes in MRSE. (a) MRSE untreated with Emilia sonchifolia. (b) MRSE treated with E. sonchifolia for 4 h. Red arrows indicate regions of the lost cellular integrity.

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TEM analysis

To examine the ultrastructural modifications in MRSE cells following treatment with E. sonchifolia, we utilized TEM. The control group exhibited intact cell walls, where in the cytoplasm remained undisturbed in conjunction with the bacterial cell membrane (Fig. 8a). Notable alterations in MRSE morphology were observed following the interaction with E. sonchifolia. Figure 8b shows the occurrence of cell wall lysis and the presence of a discontinuous cell membrane, resulting in the release of numerous intracellular components from MRSE cells. Moreover, a small proportion of the cells underwent dissolution.

Fig. 8
figure 8

Transmission electron microscopy observations of morphological changes in MRSE. (a) MRSE untreated with E. sonchifolia. (b) MRSE treated with E. sonchifolia for 4 h. Red arrows indicate cell walls. Green arrows indicate cell membranes. Yellow arrows indicate the cytoplasmic contents. Data are presented as mean (± SD) of three replicates (compared with the control, **p < 0.01).

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Discussion

S. epidermidis is one of the leading causes of BSIs35. The emergence of antibiotic-resistant strains, particularly MRSE, poses a significant challenge in the treatment of BSI. TCM posits that “clearing heat and detoxifying” TCM, which is cold in nature, can alleviate or eliminate infection caused by pathogenic microorganisms such as bacteria and viruses36. Pharmacological studies have demonstrated that clearing heat and detoxifying TCM, such as Lonicera japonica Thunb., Forsythia suspensa, Scutellaria baicalensis Georgi, and Coptis chinensis Franch. exhibit good antibacterial activity37. Similarly, in our previous study, Patrinia scabiosaefolia, classifie as a “clearing heat and detoxifying” TCM showed strong antibacterial activity against MRSE20. Additionally, a previous study revealed that E. sonchifolia extracts demonstrated strong antibacterial effects against S. aureus, P. aeruginosa and N. gonorrhoeae21. Therefore, the present study examined the in vitro and in vivo antibacterial activities of E. Sonchifolia against MRSE.

The MIC and MBC results demonstrated that E. Sonchifolia exhibits strong antibacterial efficacy in vitro, as presented in Table 1. To assess its antibacterial activity in vivo, the mouse model of MRSE-induced BSIs was established. As S. epidermidis is an opportunistic pathogen, the host immune response can eliminate it. Therefore, MRSE infections may occur in immunosuppressed patients. Based on these findings, a an immunosuppressive mouse model was developed. Subsequently, E. Sonchifolia was tested to evaluate its therapeutic efficacy against BSIs in mice. The results revealed that E. sonchifolia exhibited good antibacterial activity in vivo (Fig. 1). These findings confirm that E. sonchifolia possesses good antimicrobial activity.

Recently, a deeper understanding of antibacterial mechanisms has necessitated the integration of multi-omics data38. Proteomic and transcriptomic analyses revealed that proteins involved in purine metabolism were notably downregulated in the energy metabolic pathway. Purine metabolites play a vital role in bacterial growth39,40. In this study, proteomics and transcriptomics analyses were performed on MRSE cultured with E sonchifolia. The results indicated that proteins associated with purine metabolism were notably downregulated. These findings align with those of a previous report showing that tannic acid downregulated purine metabolism proteins in S. aureus26.

The downregulation of purine metabolism suggests inhibition of bacterial growth, providing evidence that E. sonchifolia exhibits antibacterial properties by inhibiting purine metabolism.

Purine metabolism involves metabolic pathways, such as synthesis, salvage, and regulation of purine synthesis41. The key components of purine metabolism include the aspartate and glutamate pathways. The aspartate pathway facilitates the synthesis of inosine monophosphate (IMP) from aspartic acid, followed by the synthesis of other nucleotides from IMP. Conversely, glutamate pathway involves the synthesis of GMP (guanine monophosphate) from glutamate, followed by the synthesis of other nucleotides from GMP. The purine nucleotide salvage pathway is primarily facilitated by three enzymes: adenosine kinase (ADK), adenine phosphoribosyl transferase (APRT), and hypoxanthine–guanine phosphoribosyl transferase (HGPRT). The complementary salvage pathway generally fulfills the cellular requirements for purines42. In the guanine nucleotide pathway, two enzymes facilitate the conversion of IMP to GMP43. Purines are essential organic compounds in bacterial cells, serving as fundamental components of nucleic acids, including DNA and RNA. They are integral to storage and dissemination of genetic material26. Additionally, purines play a role in bacterial energy metabolism by producing Adenosine triphosphate (ATP), that is essential for sustaining biological activities44. Studies have demonstrated that IMP is an important intermediate in de novo purine biosynthesis, because it can be converted to either GMP or AMP (Adenosine monophosphate) via two parallel pathways. The enzymes involved in the GMP pathway include IMP dehydrogenase (IMPDH) and GMP synthase (GMPS)45. Thus, in this study, the activities of GMP, AMP, and IMP were examined. Figure 6 demonstrates that E. sonchifolia effectively inhibited the activities of GMP, AMP, and IMP. These findings align with previous research46,47.

Research has revealed that inhibition of three enzymes, GMP, AMP, and IMP, correlates with the inhibition of bacterial growth ( Fig. 9). Experimental results confirmed that the activities of these enzymes were downregulated, validating that E. sonchifolia inhibits the three enzymes involved in both the synthesis and salvage pathways, thereby inhibiting purine metabolism and exerting an antibacterial effect.

Fig. 9
figure 9

Mechanistic hypothesis diagram of the purine metabolism mechanism of E. sonchifolia.

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The regulatory mechanism of bacterial purine metabolism relies on a TCS that precisely modulates the expression of pertinent genes in response to purine levels, ensuring coordinated regulation of the entire purine metabolic pathway, is the primary signal transduction system in bacteria. When external stimuli diminish, bacteria experience stress, leading to downregulation of corresponding proteins.. Proteomics and transcriptomic analyses revealed that E. sonchifolia treatment downregulated dltA proteins, that are involved in the TCS in bacterial cell wall synthesis. The cell wall and membrane serve as physical barriers, separating the internal cellular environment from the external surroundings and preventing the loss of intracellular components48. As presented in Figs. 1 and 2, E. sonchifolia disrupted the cell walls and membranes of MRSE, thereby compromising the stability of the bacterial internal environment. Similar outcomes were observed with leaf extracts of Aronia melanocarpa (Michx.), Chaenomeles superba Lindl., and Cornus mas L. tested against bacterial cells49.

Some studies have indicated that the utilization of AMP, IMP, and GMP can lead to increased levels of Uric acid , the final product of the purine metabolism pathway and is associated with increased oxidative stress50,51. We further examined changes in the defense mechanisms of MRSE. Disruption of microorganism homeostasis caused by an oxidative burst of ROS is counteracted by the cellular defense mechanisms of the antioxidant enzyme system52. This system comprises various antioxidant enzymes, including SOD, CAT, and glutathione peroxidase (GSH-Px)53. As presented in Fig. 6, the activity of SOD and CAT decreased following treatment with E. sonchifolia, demonstrating E. sonchifolia can inhibit the defense mechanisms of MRSE. Similarly, curcumin-based photodynamic inactivation can amplify oxidative damage in bacteria by inhibiting SOD and CAT activity52.

Data exhibited that E. sonchifolia inhibits both the purine metabolic synthesis and salvage pathways by inhibiting AMP, IMP, and GMP, leading to enhanced oxidative stress and ultimately exerts an antibacterial effect.

Compared with conventional antibiotics, E. sonchifolia exhibits the advantage of targeting multiple sites against MRSE. This may account for the reduced likelihood of drug resistance development in TCM. This investigation provides a preliminary elucidation of the antibacterial activity and mechanisms of E. sonchifolia against MRSE, by inhibiting purine metabolism, thereby affecting oxidative stress and damaging cell membranes and cell walls. However, further research is warranted to comprehensively understand the specific mechanism of action of E. sonchifolia against MRSE.

Conclusion

In conclusion, this study demonstrated that E. sonchifolia exhibits noteworthy antibacterial efficacy against MRSE both in vitro and in vivo. Furthermore, proteomic and transcriptome analyses predicted several antibacterial mechanisms, including inhibition of purine metabolism, increase in bacterial oxidative stress, and inhibition bacterial cell wall synthesis. Furthermore, enzymatic assays related to purine metabolism, antioxidant activity, and cell morphology analyses confirmed E. sonchifolia inhibits purine metabolism, induces oxidative stress and damages the bacterial cell wall. These findings suggest that E. sonchifolia holds great potential as an antibacterial agent.