Introduction

In recent years, the widespread use of antibiotics in aquaculture and medical practices has led to a gradual increase in antibiotic levels in the environment1,2,3. High concentrations of antibiotics exert significant inhibitory effects on microorganisms in wastewater biological treatment systems by inhibiting protein synthesis and disrupting nucleic acid formation4,5,6,7. This poses a significant challenge to wastewater treatment. Microorganisms develop resistance genes under long-term exposure to antibiotics8,9. The resistance genes persist in the environment, and spread and diffuse in different environmental media, posing greater environmental risks than the antibiotics themselves10,11,12,13. Therefore, resistance genes have garnered increasing attention as a new type of pollutant in the field of environmental pollution research in recent years. However, limited research exists on resistance genes in water environments, especially in wastewater biological treatment systems.

Plant-derived compounds have shown potential advantages of high efficiency and low toxicity, combating bacterial resistance through dual mechanisms: eliminating resistant plasmids to block genetic transmission, and disrupting physiological barriers via enhanced membrane permeability, efflux pump inhibition, and target modification, and gradually becomes a research hotspot in the process of reversing intracellular resistance genes14,15,16,17. Studies have reported that extracts from Scutellaria baicalensis can influence the expression levels of different types of proteins and genes in cells to reverse drug resistance in cancer cells, possibly through modulating NF-κB signaling pathways and downregulating efflux pump proteins18,19,20. The extracts from Folium Artemisiae Argyi can intervene in the expression of resistant genes in liver cancer cells via epigenetic modifications such as DNA methylation and histone acetylation21. The compounds including emodin can prevent the spread and dissemination of tetracycline-resistant genes in intestinal microorganisms by inhibiting plasmid conjugation efficiency through blocking type IV secretion system22. However, there is relatively limited research on the impact of specific plant extracts on protein and resistance genes during biological treatment systems. Crucially, whether such extracts can mitigate antibiotic-driven resistance gene proliferation in functional wastewater microorganisms (e.g., phosphate-accumulating organisms) remains unexplored, representing a critical gap in environmental resistance control. The study of the effects of plant extracts on the resistance genes of the main functional microorganisms in biological treatment is significant for ecological environment safety.

Biological phosphorus removal is one of the main methods for wastewater biological treatment. To address this gap, in this experiment, Exiguobacterium sp. was used as the experimental species, which is a type of polyphosphate accumulating organism. The gentamicin was selected as an antibiotic for its wide applicability and strong heat stability23. The study investigated the impact of gentamicin on the proteins and acquisition of resistance genes in Exiguobacterium sp. Subsequently, extracts from three plants (Folium Artemisiae Argyi, Scutellaria baicalensis, and Galla Chinensis) selected based on their documented traditional applications were used to research the effects on the proteins and resistance genes of Exiguobacterium sp. Raman spectroscopy was employed to observe changes in protein peaks, followed by quantitative detection of the abundance of gentamicin resistance genes in phosphate accumulating organisms. Variance analysis and Duncan’s test were conducted to analyze the mutual influence between the extracts of Folium Artemisiae Argyi, Scutellaria baicalensis, Galla Chinensis, and gentamicin resistance genes. This study is expected to contribute to a foundational theoretical basis for further research on the removal of resistance genes in the ecological environment.

Results and discussion

Changes of the protein concentration

The changes of the protein concentration for Exiguobacterium sp. are showed in Fig. 1. as can be seen in Fig. 1, under the influence of gentamicin, the protein concentration of normal Exiguobacterium sp. gradually increases with time. Around 40 min, there is a sudden sharp rise, and then stabile at 92.65 mg/L at around 110 min. The reason was that the gentamicin caused to complete leakage of the protein of the normal Exiguobacterium sp. cells. In contrast, Exiguobacterium sp. carrying the gentamicin resistance gene exhibits a consistently lower protein leakage level, stabilizing around 8 mg/L with time. This phenomenon may be attributed to the role of the gentamicin resistance gene, which facilitates the expulsion of gentamicin molecules that enter the Exiguobacterium sp. cells via a coordinated protein pump mechanism, thereby safeguarding the integrity of cell structure and function. Any minimal leakage observed is likely due to mechanical pressure during centrifugation.

Following the action of Exiguobacterium sp. with extracts from the three plants, Scutellaria baicalensis, Folium Artemisiae Argyi, and Galla Chinensis, the protein concentration gradually increased with time. Notably, after the action, Scutellaria baicalensis demonstrates a sharp increase in protein leakage trend at 40 min, stabilizing at 91.55 mg/L at 110 min; Galla Chinensis shows an escalating trend in protein leakage at 40 min, stabilizing around 88.22 mg/L at approximately 100 min; whereas Folium Artemisiae Argyi exhibits a gentle upward trend with no sudden spikes, ultimately stabilizing at 81.84 mg/L. In comparison to Folium Artemisiae Argyi and Galla Chinensis, the protein leakage situation of Exiguobacterium sp. after action with Scutellaria baicalensis closely resembles that of normal Exiguobacterium sp., indicating that Scutellaria baicalensis has the most effective action on Exiguobacterium sp. This aligns with its known efflux pump inhibition mechanism18,19,20, which disrupts gentamicin expulsion and exacerbates protein leakage.

Fig. 1
Fig. 1
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Changes of the protein concentration after the Action of Chinese Medicine(40-min leakage onset; 110-min stabilization; leakage thresholds: Scutellaria baicalensis: 91.55 mg/L; Galla Chinensis: 88.22 mg/L; Folium Artemisiae Argyi: 81.84 mg/L).

Changes in Raman spectra of protein after the action of Scutellaria baicalensis, Folium Artemisiae Argyi, and Galla Chinensis

The Raman Spectra of protein, after the action of Scutellaria baicalensis and Galla Chinensis, are depicted in Fig. 2. In Fig. 2, it is evident that there are some differences in the Raman Spectra of the protein of Exiguobacterium sp. after the action of Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis.

Fig. 2
Fig. 2
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Raman Spectral Changes of Proteins After action by Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis ((a) Scutellaria baicalensis induces C=C bond instability (1543-1585 cm⁻¹), (b) Folium Artemisiae Argyi enhances C-H bond rigidity (1460-1520 cm⁻¹), (c) Galla Chinensis triggers low-energy bond cleavage (1425-1780 cm⁻¹). Highlighted peaks correlate with gentamicin resistance modulation mediated by Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis.)

During the action process with Scutellaria baicalensis extract, the prominent characteristic peaks of Raman Spectra for control group and experimental group are located at 1543 cm− 1, 1553 cm− 1, 1445 cm− 1, and 1585 cm− 1 in the normal and reversed Raman Spectra of Exiguobacterium sp. The peaks at 1543 cm− 1, 1585 cm− 1, and 1553 cm− 1 correspond to unsaturated carbon (C = C) bonds, and the peak at 1445 cm− 1 corresponds to the CH2 wagging vibration (mainly C3H6). This suggests longer bond lengths, lower bond energies, and unstable bond positions, leading to susceptibility to external intrusion by gentamicin and subsequent bond breakage.

During the action process with Folium Artemisiae Argyi extract, the prominent characteristic peaks of Raman Spectra for control group and experimental group exhibit strong and conspicuous similarities, with characteristic peaks located predominantly at 1460 cm− 1, 1475 cm− 1, and 1520 cm− 1. Specifically, the peak at 1460 cm− 1 corresponds to CH2 rocking vibrations (primarily C5H10), the peak at 1475 cm− 1 corresponds to CH2 scissoring vibrations, and the peak at 1520 cm− 1 corresponds to three H bonds of amide groups. In the reversed Exiguobacterium sp., Raman Spectra of proteins display additional peaks at 2080 cm− 1 and 2133 cm− 1. The peak at 2080 cm− 1 corresponds to symmetric vibrations of C = C = C = C, while the peak at 2133 cm− 1 corresponds to H bonds containing Si and Ar. These bonds, relative to C-H bonds, exhibit high stability, albeit with low peak intensity, possibly due to the low absorption efficiency of Folium Artemisiae Argyi by the bacterial strain, leading to the presence of unreversed cells. The observed C = C = C = C bond stabilization (880 cm⁻¹) directly correlates with plant extract-induced target modification mechanisms described in prior studies14,15,16,17.

During the action process with Galla Chinensis extract, the prominent characteristic peaks of Raman Spectra for control group and experimental group are located at 1425 cm− 1, 1445 cm− 1, 1550 cm− 1, 1565 cm− 1, and 1780 cm− 1. Specifically, the peak at 1425 cm− 1 corresponds to the anti-symmetric oscillation of C-H in CH3-C = O, the peak at 1445 cm− 1 corresponds to CH2 rocking vibrations (primarily from C3H6), the peak at 1550 cm− 1 corresponds to unsaturated bonds of C, typically containing Br or Cl, the peak at 1565 cm− 1 corresponds to H bonds of olefins, and the peak at 1780 cm− 1 corresponds to H bonds in (C3H4) = CH2. These bond positions have relatively low bond energies. They are prone to breakage, indicating that the intracellular proteins of both normal and reversed Exiguobacterium sp. are unable to withstand the invasion of gentamicin.

The characteristic peaks in the Spectra of Exiguobacterium sp. containing gentamicin resistance genes are located at 829 cm− 1, 880 cm− 1, 942 cm− 1, 1005 cm− 1, 1025 cm− 1, and 1445 cm− 1. Among these, 829 cm− 1 corresponds to the out-of-plane vibration of C-H in H-C = C-H, 880 cm− 1 corresponds to the symmetric vibration of C = C = C = C, 942 cm− 1 corresponds to the stretching vibration of C = C and C-O, 1005 cm− 1 corresponds to the stretching vibration of C = C, 1025 cm− 1 corresponds to the in-plane bending vibration of C = C = C and the swinging vibration of O-H, and 1445 cm− 1 corresponds to the rocking vibration of CH2 (mainly from C3H6). However, the intensity of the peak at 1445 cm− 1 is low, indicating a lower content of this component. Compared to other unsaturated bonds such as C = C, C-O, C = C = C, C = C = C = C, and C-H bonds, which have relatively shorter bond lengths and higher bond energies, providing stability, this peak may play a role in transporting gentamicin molecules and binding responsive proteins, facilitating the expulsion of intracellular gentamicin through the cell membrane pump when facing external gentamicin pressure. The lower intensity of the peak at 942 cm− 1 compared to other C = C positions suggests that this may be due to the surface selection rule, where chemical bonds perpendicular to the substrate can be significantly enhanced, while those parallel to the substrate are weaker. Therefore, this position may not be entirely perpendicular to the silver nano-substrate but tilted to the substrate surface, undergoing adsorption by Ag particles, consistent with the phenomenon and mechanism described by Han et al., where Ag substrates can adsorb near carboxyl groups (—COO—).

Changes in the abundance of Gentamicin Resistance Genes after the action of Scutellaria baicalensis, Folium Artemisiae Argyi, and Galla Chinensis

The changes in the abundance of gentamicin resistance genes in Exiguobacterium sp. before and after the administration of extracts from Scutellaria baicalensis, Folium Artemisiae Argyi, and Galla Chinensis are illustrated in Fig. 3. All data were analyzed using one-way analysis of variance and Duncan’s test, with P values consistently below 0.05, indicating statistical significance. As shown in Fig. 3, the intracellular abundance of resistance genes in normal Exiguobacterium sp. is relatively low, around 0.01 copies/g, with the highest content of rmtB reaching 0.023 copies/g. This suggests that the inability of normal Exiguobacterium sp. to resist gentamicin invasion is due to its low intracellular abundance of gentamicin resistance genes.

Fig. 3
Fig. 3
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The changes in resistance gene abundance before and after the action by Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis. Different superscript letters indicate statistically significant differences among samples (Duncan’s test, P < 0.05). Asterisks (*) denote correlations significant at P < 0.01 level.(Galla Chinensis reduced rmtD to 1.5 copies/g, while Scutellaria baicalensis showed the weakest rmtA suppression.)

After long-term exposure to gentamicin, there was a significant increase in the intracellular abundance of gentamicin resistance genes in Exiguobacterium sp. According to the reference primer sequences in Table 1, the abundance of armA gene was 2.57 × 1010 copies/g, rmtB gene was 7.68 × 109 copies/g, rmtA gene was 1.15 × 1010 copies/g, rmtC gene was 1.41 × 1010 copies/g, and rmtD gene was 5.22 × 109 copies/g. This indicates a notable enhancement in the intracellular abundance of gentamicin resistance genes compared to normal Exiguobacterium sp. previously observed. When facing gentamicin invasion, the bacterial cells utilize synthesized carrier proteins to pump out gentamicin molecules through the cell membrane, thereby protecting the structural and functional integrity of the cells. This mechanism of efflux pump-mediated resistance is similar to the findings of Liu et al.24.

After treatment with Scutellaria baicalensis, Folium Artemisiae Argyi, and Galla Chinensis, the intracellular abundance of gentamicin resistance genes in Exiguobacterium sp. significantly decreased. Following Scutellaria baicalensis treatment, the abundance of armA was 72.3 copies/g, rmtB was 81.3 copies/g, rmtA was 157 copies/g, rmtC was 34.1 copies/g, and rmtD was 598 copies/g. Previous studies have indicated that during the action process with Scutellaria baicalensis, its active components can inhibit the expression of certain genes, thereby suppressing the production of transport proteins, allowing gentamicin to enter cells without being affected by efflux, and reaching binding sites to exert its bactericidal effect25.

After treatment with Folium Artemisiae Argyi, the abundance of armA was 72.3 copies/g, rmtB was 81.3 copies/g, rmtA was 157 copies/g, rmtC was 34.1 copies/g, and rmtD was 598 copies/g; the reversal effect was significant, although the mechanism remains unknown.

After treatment with Galla Chinensis, the abundance of armA was 26.3 copies/g, rmtB was 5.37 copies/g, rmtA was 15 copies/g, rmtC was 10.9 copies/g, and rmtD was 1.5 copies/g. This aligns with findings from Zhai26 and others, suggesting that the effective components of Galla Chinensis can inhibit the expression of certain mRNA, thereby suppressing the expression of resistance genes. The drastic reduction in rmtD abundance (to 1.5 copies/g) reflects the plasmid elimination effects mediated by the phytochemicals derived from Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis22, confirming their dual genetic-physiological intervention mode.

Potential role of Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis and their bioactive components in reversing gentamicin resistance by bacteria

Several bioactive components within Scutellaria baicalensis, Folium Artemisiae argyi, and Galla Chinensis have been identified as potential contributors to the observed gentamicin resistance reversal and their ability to synergize with gentamicin. When gentamicin acts on cells, the cells stimulate the expression of stress proteins through self-protective mechanisms, thereby developing resistance to gentamicin. Studies have shown that the main components of Scutellaria baicalensis and Folium Artemisiae argyi are flavonoids, which can downregulate the expression levels of stress proteins, reducing or even eliminating drug resistance27,28. Additionally, research indicates that the main component of Galla Chinensis is tannins, which can inhibit the expression of stress proteins29. Following treatment with Scutellaria baicalensis, Folium Artemisiae argyi, or Galla Chinensis, the downregulation of stress protein expression reduces bacterial resistance to gentamicin.

Methods

Bacterial source

The phosphate-accumulating organisms used in the experiment were Exiguobacterium sp., which was cultivated and preserved by the Anhui Provincial Key Laboratory of Water Pollution Control and Wastewater Resource Utilization at Anhui Jianzhu University, with a phosphate removal rate of 95.12%. The phosphate removal rate of 95.12% was determined through laboratory performance testing: the initial phosphorus concentration in the culture medium was 10.31 mg/L, and the effluent phosphorus concentration of the optimal strain reached 0.47 mg/L, resulting in a calculated removal efficiency of 95.12%.

Phosphorus removal efficiency testing

The phosphorus removal capacity of Exiguobacterium sp. was evaluated using synthetic wastewater containing 10.31 mg/L of phosphorus (as KH2PO4). After 24-hour incubation under optimal growth conditions (30 °C, pH 7.2), the residual phosphorus concentration in the supernatant was measured by the molybdenum blue method (Standard Methods 4500-P E). Removal efficiency (η, %) was calculated as:

η = [(C0 - Ce)/C0] × 100%.

where C0 and Ce represent the initial and final phosphorus concentrations (mg/L), respectively.

Drug reagents

The extracts of Folium Artemisiae Argyi, Scutellaria baicalensis, and Galla Chinensis were all sourced from Shaanxi Ruikang Biotechnology. The concentrations of the herbal medicines were set to half of the minimum inhibitory concentration (MIC) determined through preliminary experiments. Briefly, MIC values were measured by the broth microdilution method: Exiguobacterium sp. was incubated with serially diluted herbal extracts in LB medium at 30 °C for 24 h, and the MIC was defined as the lowest concentration showing complete growth inhibition. Based on these measurements, the applied concentrations were: Scutellaria baicalensis 1.17 g/L (1/2 MIC = 2.34 g/L), Galla Chinensis 0.55 g/L (1/2 MIC = 1.10 g/L), and Folium Artemisiae Argyi 2.42 g/L (1/2 MIC = 4.84 g/L). The protein extraction kit, BCA assay kit, DNA extraction and quantification kit, T vector and ligation kit, competent cell preparation kit, PCR amplification kit, agarose gel DNA recovery kit, M13 vector and amplification kit, etc., were purchased from Shanghai Simgen.

PCR conditions

PCR amplification was performed in a reaction mixture containing 1 µL of genomic DNA or lysate, 25 µL of Taq PCR Master Mix, and 2 µL of target-specific primers. The thermal cycling protocol consisted of an initial denaturation at 96 °C for 5 min, followed by 40 cycles of denaturation (96 °C for 30 s), annealing (55 °C for 30 s), and extension (72 °C for 1 min), with a final extension step at 72 °C for 10 min. Post-amplification, the PCR products were analyzed by electrophoresis on a 2% agarose gel. The primers required were synthesized by Nanjing Kingsray Biotech Co., Ltd. The specific primers are listed in Table 1.

Table 1 Primer sequence and length.
Full size table

Test methods

Protein and DNA extraction and determination

The intracellular protein extraction of Exiguobacterium sp. in this experiment was conducted using a total protein extraction kit. Concentration measurement of protein was performed using the BCA assay kit. DNA extraction and concentration measurement were both carried out using specific assay kits.

Detection of protein and resistance gene abundance

The herbal extract solution was diluted to half the minimum inhibitory concentration (1/2 MIC) and uniformly coated onto LB agar plates. Exiguobacterium sp. was inoculated into 30 mL of LB liquid medium and cultured in a shaking incubator at 37 °C with agitation (125 rpm) for 24 h. Subsequently, the bacterial cells were harvested via centrifugation, washed, and resuspended in 30 mL of 20 mmol/L phosphate-buffered saline (PBS, pH 7.0). Protein content of the solution was quantitatively assessed at 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min intervals. The protein concentration was determined using the BCA assay kit with Raman spectroscopy detection, was found to be 1 g/L. Protein Raman Spectra were collected using a laser confocal Raman spectrometer (model CRM 2000, USA), with data acquisition at a wavelength of 532 nm. The abundance of gentamicin resistance genes was assessed through agarose gel electrophoresis to confirm the presence of five resistance genes (armA, rmtB, rmtA, rmtC, rmtD). Gel fragments containing the identified genes were recovered, followed by gentamicin resistance gene T-vector transformation, gene cloning, and M13 amplification. Subsequent confirmation of successful gene transformation led to the purification of gentamicin resistance gene standards. These standards were diluted 10^5 to 10^9 times and used as templates to construct a standard curve on a fluorescence quantitative PCR instrument (model Applied Biosystems-7500, USA). All samples were run in triplicate, and the average values were calculated. The standard curve in the experiment exhibited a correlation coefficient (R2) ranging from 0.98 to 0.99. The experimental procedures closely followed the instructions provided in the assay kit manual.

Data analysis

The data obtained from the experiment were subjected to one-way analysis of variance (one-way ANOVA, Duncan’s test) using SPSS 21.0 software, and graphical representations were generated using Origin software.

Conclusions

Compared to normal Exiguobacterium sp., there was no significant change in the phosphate removal rate of gentamicin-resistant Exiguobacterium sp. after the action of Chinese medicine extract. However, relative to Folium Artemisiae Argyi and Galla Chinensis, Scutellaria baicalensis has the best effect on Exiguobacterium sp. in terms of reducing resistance genes. According to the Raman Spectra test results, the similarity in protein between post-treated resistant Exiguobacterium sp. and normal strains was relatively high, indicating a favorable reversal effect. Resistance gene abundance testing showed a significant decrease in intracellular gentamicin resistance gene abundance after herbal treatment. These research findings will provide foundational theoretical support for further in-depth studies on removing resistance genes in the ecological environment. The study also discussed the potential roles of key bioactive components: flavonoids in Scutellaria baicalensis and Folium Artemisiae argyi, and tannins in Galla Chinensis.