Use of astaxanthin in inhibiting conjugative transfer of antibiotic resistance genes, methods and a bacterio-algal symbiotic system

Abstract

This application belongs to the interdisciplinary field of biomedicine and environmental water treatment, specifically relating to the use, method, and algal-microbe symbiotic system of astaxanthin in inhibiting the conjugation and transfer of antibiotic resistance genes. This invention involves adding astaxanthin to the environment to maintain its effective free concentration in the aqueous phase at 1–20 μg / L. When using an algal-microbe symbiotic sludge treatment system, Haematococcus pluvialis continuously releases astaxanthin in situ within the system, maintaining the effective free concentration in the aqueous phase at 1–20 μg / L, thus achieving continuous inhibition without frequent external addition. This invention also provides a corresponding algal-microbe symbiotic sludge treatment system that utilizes the in-situ continuous release of astaxanthin from Haematococcus pluvialis, suitable for treating municipal sewage, hospital wastewater, and aquaculture wastewater. This invention requires no external chemical agents, is environmentally friendly, and can inhibit the spread of antibiotic resistance genes at the source.

Description

Technical Field

[0001] This invention relates to the interdisciplinary fields of biomedicine and environmental water treatment, specifically to the use, methods, and algal symbiotic systems of astaxanthin in inhibiting the conjugation and transfer of antibiotic resistance genes. Background Technology

[0002] The mass production and widespread use of antibiotics have fueled the continued spread of antibiotic resistance, posing a significant challenge to global public health. Antibiotic resistance genes (ARGs), serving as the genetic basis of bacterial resistance, can be stably passed down through vertical inheritance and rapidly spread between different microbial species via horizontal gene transfer (HGT). This promotes the formation and spread of resistant strains, increasing environmental exposure risks and the difficulty of treating clinical infections.

[0003] Wastewater treatment plants collect antibiotics, drug-resistant bacteria, resistance genes, and various mobile genetic elements (MGEs) from domestic sewage, hospital wastewater, livestock and poultry wastewater, and industrial wastewater, and are considered important reservoirs and diffusion hotspots for antibiotic resistance genes (ARGs) in the environment. Numerous studies have shown that while traditional activated sludge treatment systems can effectively remove organic pollutants and nutrients, their control of ARGs is limited. Some ARGs can still enter the natural environment with the effluent or residual sludge, further increasing the risk of antibiotic resistance pollution.

[0004] Existing technologies for controlling ARGs mainly include physicochemical methods such as ozone oxidation, ultraviolet disinfection, advanced oxidation, electrochemical oxidation, membrane separation, and adsorption. These methods can reduce the number of resistant bacteria or degrade free DNA to some extent, but they generally suffer from problems such as high equipment investment, high energy consumption, high treatment costs, easy formation of disinfection byproducts, and insufficient long-term stability. Furthermore, most existing technologies primarily target the removal of ARGs themselves, without effectively controlling the key processes of ARG transmission, thus failing to fundamentally reduce the environmental transmission risk of drug-resistant genes.

[0005] Existing research has mainly focused on indirectly weakening the spread of ARGs by reducing bacterial numbers, degrading DNA, or altering the structure of microbial communities. However, there is still a lack of green, efficient, and suitable technical solutions for the long-term operation of wastewater treatment systems to directly reduce bacterial conjugation and transfer capabilities, inhibit the activation of mobile genetic elements, and reduce horizontal gene transfer efficiency.

[0006] In recent years, some studies have begun to focus on direct inhibition strategies for conjugation transfer, such as the application exploration of baicalin and ε-polylysine. Baicalin can effectively inhibit the conjugation transfer of RP4 plasmids by binding to LsrB protein and interfering with the AI-2 quorum sensing system, within a concentration range of 12.5–100 μg / mL. ε-polylysine, on the other hand, reduces cell surface hydrophobicity and induces oxidative stress, not only downregulating conjugation-related genes such as trfAp and trbBp, but also upregulating the expression of global repressor genes such as trbA, korA, and korB, showing a significant inhibitory effect even at a concentration of 10 mg / L. However, the effective concentrations of these inhibitors are still in the milligrams per liter range, far exceeding the acceptable dosage levels for practical wastewater treatment, and most studies remain at the laboratory stage, without achieving green, low-concentration, and sustainable engineering applications. Summary of the Invention

[0007] The purpose of this invention is to overcome the problem in the prior art of lacking a green, efficient method suitable for long-term operation of wastewater treatment systems, capable of directly reducing bacterial conjugation and transfer capabilities, inhibiting the activation of mobile genetic elements, and reducing horizontal gene transfer efficiency. This invention provides the use, method, and bacterial-algal symbiotic system of astaxanthin in inhibiting antibiotic resistance gene conjugation and transfer. This invention discovers that astaxanthin, by reducing intracellular reactive oxygen species levels in bacteria and downregulating the expression of conjugation and transfer-related genes, directly acts on the RP4 plasmid-mediated conjugation and transfer process, effectively reducing horizontal gene transfer efficiency.

[0008] It should be noted that the effective concentration described in this invention refers to the actual detectable concentration of free astaxanthin in the aqueous phase. In the algal-bacterial symbiotic system, astaxanthin released in situ by Haematococcus pluvialis exists in the aqueous phase in a free form, and its effective concentration is maintained within the activity range of 1–20 μg / L through continuous release. In the examples, the measured total astaxanthin content (including intracellular and bound forms) was significantly higher than the effective concentration of free astaxanthin in the aqueous phase.

[0009] A first aspect of the invention provides the use of astaxanthin in the preparation of inhibitors for inhibiting antibiotic resistance gene conjugation transfer mediated by the RP4 plasmid. Preferably, the astaxanthin is obtained from Haematococcus pluvialis.

[0010] Furthermore, the concentration of astaxanthin in the inhibitor is 1–20 μg / L. Preferably, the concentration of astaxanthin in the inhibitor is 1–10 μg / L.

[0011] Furthermore, the inhibitor inhibits antibiotic resistance gene conjugation transfer mediated by the RP4 plasmid by reducing intracellular reactive oxygen species levels in bacteria and downregulating the expression of conjugation transfer-related genes.

[0012] Furthermore, the inhibitor is used in wastewater treatment systems to suppress the spread of antibiotic resistance genes.

[0013] Furthermore, the astaxanthin is produced in situ by Haematococcus pluvialis in a symbiotic sludge treatment system.

[0014] Furthermore, in the aforementioned algae-bacteria coexisting sludge treatment system, the dry weight-based algae-to-sludge ratio of Haematococcus pluvialis to activated sludge in the reactor is 1:2 to 1:8 (w / w); the dry weight refers to the mass of the sample after drying at 105°C to constant weight, with a light intensity of 1000 to 5000 lux and dissolved oxygen maintained at 2 to 4 mg / L.

[0015] A second aspect of the present invention provides a method for inhibiting antibiotic resistance gene conjugation transfer mediated by RP4 plasmid, comprising: adding astaxanthin to an environment containing a target microorganism, thereby maintaining the astaxanthin concentration in the environment at 1–20 μg / L.

[0016] Preferably, astaxanthin released by Haematococcus pluvialis is added to an environment containing the target microorganism, so that the effective concentration of free astaxanthin in the environmental water phase is maintained at 1-20 μg / L.

[0017] A third aspect of the present invention provides a symbiotic sludge treatment system for inhibiting the spread of antibiotic resistance genes, comprising an activated sludge reactor and Haematococcus pluvialis added therein; wherein the ratio of Haematococcus pluvialis to activated sludge in the reactor based on dry weight is 1:2 to 1:8 (w / w); wherein the dry weight refers to the mass of the sample after drying at 105°C to constant weight, the light intensity is 1000 to 5000 lux, and the dissolved oxygen is maintained at 2 to 4 mg / L.

[0018] Preferably, the system includes an activated sludge reactor and Haematococcus pluvialis added therein; the Haematococcus pluvialis is a green vegetative cell in the logarithmic growth phase; the dry weight-based algae-to-sludge ratio of Haematococcus pluvialis to activated sludge is 1:2 to 1:8 (w / w), the light intensity is 1000 to 5000 lux, and the dissolved oxygen is maintained at 2 to 4 mg / L; the system also includes a maintenance mechanism for regularly monitoring the biomass of Haematococcus pluvialis and supplementing algal cells according to the monitoring results.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides an application of astaxanthin in the preparation of inhibitors. By using astaxanthin as the active ingredient to prepare inhibitors, astaxanthin can reduce the level of reactive oxygen species in bacterial cells and downregulate the expression of conjugation transfer-related genes, thereby inhibiting the conjugation transfer of antibiotic resistance genes mediated by RP4 plasmid. Moreover, the inhibitory effect is significantly better than that of baicalin under the same concentration conditions.

[0020] 2. This invention provides a method for inhibiting the conjugation transfer of antibiotic resistance genes mediated by RP4 plasmid. By adding astaxanthin to an environment containing the target microorganism and controlling its concentration to be maintained at 1-20 μg / L, the frequency of conjugation transfer can be significantly reduced. The method is simple to operate, highly controllable, and can be flexibly applied to different treatment scenarios.

[0021] 3. This invention provides a symbiotic sludge treatment system for bacteria and algae. By coupling an activated sludge reactor with Haematococcus pluvialis, the algae-to-sludge ratio is controlled at 1:2 to 1:8 (w / w), the light intensity is 1000 to 5000 lux, and the dissolved oxygen is 2 to 4 mg / L. Haematococcus pluvialis continuously releases natural metabolites such as astaxanthin in situ within the system, which can significantly reduce the abundance of antibiotic resistance genes and mobile genetic elements. Moreover, it requires no external chemical agents, does not produce toxic byproducts, and is compatible with existing wastewater treatment processes. Attached Figure Description

[0022] Figure 1 This is a reactor configuration diagram of the bacterial-algae symbiotic sludge treatment system in an embodiment of the present invention; Figure 2 A comparison of the abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in a traditional activated sludge system (R0) and a microbial-algae symbiotic system (R1); Figure 3 Figure showing the screening of differential metabolites and analysis of key antioxidant metabolites after treatment with Haematococcus pluvialis; Figure 4 Figure 1 shows the effect of different concentrations of astaxanthin on the binding transfer frequency of RP4 plasmid. Figure 5 Image of laser confocal microscopy for bonding transfer; Figure 6 A comparison chart showing the efficiency of different treatments in scavenging reactive oxygen species (ROS). Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0024] Example 1 Construction and operation of fungal-algae symbiotic system like Figure 1As shown, sludge collected from the secondary sedimentation tank of a wastewater treatment plant in Qingdao was acclimated and inoculated into an SBR reactor with an initial concentration of 3000 mg / L. A microporous aeration disc with a working volume of 1.1 L was installed at the bottom of the reactor, and the pH of the reaction system was adjusted to 7.0–7.5. The reactor temperature was maintained at 20–30 °C, preferably 25 °C; the light intensity was 1000–5000 lux, preferably 1500–2000 lux, with a 12-hour light / 12-hour dark cycle simulating sunlight; dissolved oxygen (DO) was maintained at 2–4 mg / L, preferably 2.5 mg / L; the hydraulic retention time (HRT) was 12–20 h, preferably 16 h; and the algae-to-sludge ratio based on dry weight was 1:2–1:8 (w / w), preferably 1:4 (dry weight). The system operates for three cycles daily, each lasting four hours, including 5 minutes of influent, 168 minutes of aeration, 62 minutes of sedimentation, and 5 minutes of effluent. Influent and effluent are exchanged via a peristaltic pump at a 50% volumetric exchange ratio. The wastewater used in this embodiment is artificially synthesized, and the influent formulation consists of organic carbon sources CH3COONa and C6H4O2. 12 O6, total content approximately 350 mg / L; nitrogen source is NH4Cl, content 50 mg / L; phosphorus source is KH2PO4, content 5 mg / L.

[0025] To ensure the long-term stable operation of the algal symbiotic system, the biomass of Haematococcus pluvialis in the reactor needs to be monitored and maintained regularly. The monitoring method is as follows: samples are taken at fixed times daily, and the algal cell density is counted under a microscope using a hemocytometer, or measured using OD (Organic Oxygen Demand). 680 A rapid estimation is performed. When the algal cell density falls below 70% of the initial inoculum, Haematococcus pluvialis green vegetative cells in the logarithmic growth phase are added, with the addition amount aimed at restoring the algal cell density to the initial level. The addition frequency is generally once every 3 to 7 days, depending on actual operation. In addition, by controlling the hydraulic retention time (HRT) to 12 to 20 hours and the settling time to 62 minutes, flocs with good settling properties (including symbiotic flocs of microalgae and activated sludge) are retained in the reactor, and the microalgae lost with the effluent are compensated for by the above-mentioned addition strategy.

[0026] The implementation examples of this invention adopt a three-stage operation mode of stable operation, antibiotic stress, and natural metabolite intervention.

[0027] Phase 1: No antibiotics are added to the influent, the system operates stably, and a basic microbial community is established; Second stage: Add multiple antibiotics to the influent (tetracycline Tet, kanamycin sulfate Kan, and ampicillin Amp, all at 500 μg / L). Phase 3: While maintaining the antibiotic concentration from Phase 2, Haematococcus pluvialis was added to the experimental system to construct a stable symbiotic system. The Haematococcus pluvialis was in the green vegetative cell stage before inoculation and was pre-cultured in BG-11 medium at a light intensity of 2000 lux, a light-dark ratio of 12 h:12 h, and a temperature of 25°C until the logarithmic growth phase (OD). 680 The microalgae (approximately 0.6–0.8 g / L) are collected by centrifugation and added to the SBR reactor at an initial inoculum amount of 25% of the dry weight of the activated sludge in the reactor (i.e., an algae-to-sludge ratio of 1:4). During operation, the microalgae secrete metabolic products that continuously affect the system.

[0028] In the algae-bacterial symbiotic system of this invention, Haematococcus pluvialis is present in the metabolic products of activated sludge (CO2, NH4). + The algae are in a mildly induced microenvironment characterized by a combination of organic matter degradation intermediates and periodic light variations. This environment is neither the optimal growth conditions for pure culture nor a severe lethal stress such as high light intensity or nutrient starvation, but rather a continuous state of mild oxidative stress that stimulates algal cells to moderately synthesize and release astaxanthin without affecting their basic growth and metabolic activities. Turbulence generated by aeration within the reactor causes algal cells to circulate between light and dark zones. This periodic change in the light environment further constitutes a mild stress signal, promoting the sustained low-level expression and release of astaxanthin.

[0029] This implementation case uses artificial water distribution to ensure consistency of initial conditions, facilitating mechanism research. In practical engineering applications, this algal-microbe symbiotic system is also applicable to municipal sewage, hospital wastewater, aquaculture wastewater, and combinations thereof. When treating actual wastewater, those skilled in the art can adaptively adjust the following parameters according to the influent water quality: if the influent turbidity is high or the COD load is large, the hydraulic retention time can be appropriately extended to 20-30 hours, or the light intensity can be increased to 3000-5000 lux to compensate for light attenuation; if the influent contains toxic and harmful substances that may inhibit microalgae activity (such as heavy metals, residual disinfectants, etc.), it is recommended to install a pretreatment unit (such as coagulation sedimentation or activated carbon filtration) at the influent end to reduce the concentration of inhibitory substances to within the tolerance range of microalgae before entering the algal-microbe symbiotic reactor; the initial operation period (the first 7-14 days) is the microalgae adaptation period. During this period, if the algal cell density decreases, the hydraulic retention time can be temporarily shortened to 10-12 hours to improve the biomass retention rate, and then restored to the target value after the algal cell density recovers. All of the above adjustments are within the scope of the core parameters defined in the claims of this invention, and those skilled in the art can make adaptive adjustments based on specific water quality without any creative effort.

[0030] Example 2 Screening key metabolites using metabolomics Samples from the natural metabolite intervention stage of the system in Example 1 were analyzed for metabolomics using liquid chromatography-mass spectrometry (LC-MS). Differential metabolite screening was based on the VIP value in the OPLS-DA model and the p-value of Student's t-test. If the VIP value was greater than 1 and the p-values ​​after multiple test correction were less than 0.05, it was considered to be significantly different.

[0031] Results of differential metabolite screening and analysis of key antioxidant metabolites are as follows: Figure 3 As shown. By Figure 3 It was found that after the introduction of Haematococcus pluvialis in stage three, the metabolite composition of R1 differed significantly from that of R0, with 355 differentially expressed metabolites identified. Astaxanthin exhibited the highest fold change and was the main characteristic metabolite. The total astaxanthin content (including intracellular and bound forms) in the system was significantly higher than that in R0 at the same stage, with a fold increase of 5–7 times. This total content included multiple forms, including intracellular accumulation, cell-bound forms, and free forms in the aqueous phase, with the effective concentration of free forms in the aqueous phase maintained at 1–20 μg / L. Furthermore, cofactor antioxidant metabolites such as erythritol, 3-hydroxybutyric acid, yakuchinone A, parthenolide, and russselioside B were also significantly enriched in R1. Therefore, astaxanthin is an important functional metabolite in the algal-bacterial system for inhibiting the spread of ARGs.

[0032] Example 3 Astaxanthin inhibits ARGs binding transfer E. coli MG1655, carrying the RP4 plasmid, was used as the donor strain. Its chromosome was marked with the lacI^q^ and mCherry genes. This strain also carried the broad-host-specific conjugation plasmid RP4, serving as the ARGs transfer vector for the study. The RP4 plasmid used in the experiment carried a green fluorescent protein (GFP) marker gene for fluorescence tracking; this RP4 plasmid contained resistance genes against tetracycline, kanamycin, and ampicillin. In E. coli MG1655, the chromosomal lacI^q^ suppressed the expression of the GFP gene on the RP4 plasmid, thus the donor strain only exhibited red fluorescence specifically encoding the mCherry gene. However, when the RP4 plasmid was horizontally transferred into a recipient strain that did not carry the lacI^q^ gene, the marked GFP gene was activated due to the absence of lacI^q^, resulting in green fluorescence expression in the recipient strain. P. aeruginosa HLS-6, carrying a streptomycin resistance gene, was used as the recipient strain.

[0033] After centrifuging the donor and recipient bacteria separately (8000×g, 5 min) to collect the bacterial cells, they were washed with sterile phosphate buffer (pH 7.2) and resuspended to OD. 600= 0.4. The two bacterial suspensions were mixed at a 1:1 volume ratio and incubated at 30°C for 10 hours. The conjugation transfer experiment included a control group (no antibiotics), an antibiotic stress group (tetracycline, kanamycin, and ampicillin concentrations of 10 μg / L and 500 μg / L, respectively), an antibiotic + low-concentration astaxanthin treatment group (astaxanthin concentration of 1 μg / L), and an antibiotic + high-concentration astaxanthin treatment group (astaxanthin concentration of 20 μg / L).

[0034] RP4 plasmid conjugation and transfer frequencies in different treatment groups, such as Figure 4 As shown. Compared with the control group, the conjugation transfer frequency increased 5.3-fold under low-concentration antibiotic stress, while the conjugation transfer frequencies decreased by 96% and 76% in the low-concentration and high-concentration astaxanthin treatment groups, respectively (relative to the antibiotic stress group). Laser confocal results ( Figure 5 As can be seen, the number of conjugates (green cells) was significantly increased after antibiotic stress compared to the control group. Conjugation transfer was inhibited in the astaxanthin-added group, and the number of conjugates was significantly reduced. This provides direct experimental evidence for the mechanism by which microalgal metabolites intervene in antibiotic resistance genes in a microbial-algal symbiotic system.

[0035] Example 4 Astaxanthin inhibits ROS generation in ARGs conjugation and transfer systems Based on Example 3, a plasmid conjugation and transfer system was constructed, with the following groups set up: a control group, a low-concentration antibiotic supplementation group (10 μg / L Amp + 10 μg / L Kan + 10 μg / L Tet), a high-concentration antibiotic supplementation group (500 μg / L Amp + 500 μg / L Kan + 25 μg / L Tet), a low-concentration antibiotic + low-concentration astaxanthin supplementation group (10 μg / L Amp + 10 μg / L Kan + 10 μg / L Tet, astaxanthin concentration 1 μg / L), a high-concentration antibiotic + high-concentration astaxanthin supplementation group (500 μg / L Amp + 500 μg / L Kan + 25 μg / L Tet, astaxanthin concentration 20 μg / L), a low-concentration antibiotic + thiourea supplementation group (10 μg / L Amp + 10 μg / L Kan + 10 μg / L Tet, 100 μM thiourea), and a high-concentration antibiotic + thiourea supplementation group (500 μg / L Amp + 500 μg / L Kan + 25 μg / L Tet, astaxanthin concentration 1 μg / L), and a high-concentration antibiotic + thiourea supplementation group (500 μg / L Amp + 500 μg / L Kan + 25 μg / L Tet, astaxanthin concentration 20 μg / L), a low-concentration antibiotic + thiourea supplementation group (10 μg / L Amp + 10 μg / L Kan + 10 μg / L Tet, 100 μM thiourea), and a high-concentration antibiotic + thiourea supplementation group (500 μg / L Amp + 500 μg / L Kan + 2 The ROS content in the system was determined using the DCFH-DA fluorescent probe method. The mixed bacterial cells of the conjugation transfer system were washed with PBS buffer and resuspended, then DCFH-DA working solution (final concentration 10 μmol / L) was added and incubated at 37 ℃ in the dark for 30 min. After washing away probes that did not enter the cells, the fluorescence intensity was measured using a fluorescent microplate reader (Ex / Em = 488 / 525 nm).

[0036] The results of ROS content changes in different treatment groups are as follows: Figure 6 As shown in the figure, in the control group and the low / high concentration antibiotic addition groups, the ROS scavenging rates of low concentration astaxanthin were 14.1%, 66.4%, and 30.9%, respectively; while the ROS scavenging rates of thiourea in the corresponding treatment groups were 12.6%, 35.7%, and 26.0%. The results indicate that astaxanthin has a superior ROS scavenging ability for the ARGs conjugation transfer system, thereby inhibiting the horizontal gene transfer of ARGs.

[0037] Example 5 Comparative experiment on the inhibition of RP4 plasmid binding and transfer by astaxanthin and baicalin 5.1 Experimental Materials Donor strain: Escherichia coli MG1655 carrying the RP4 plasmid, whose chromosome is marked with the lacI^q^ and mCherry genes; the RP4 plasmid carries the green fluorescent protein (GFP) marker gene and tetracycline, kanamycin and ampicillin resistance genes.

[0038] Recipient strain: Pseudomonas aeruginosa HLS-6, carrying the streptomycin resistance gene.

[0039] Test substances: Astaxanthin standard (purity ≥98%), baicalin standard (purity ≥98%).

[0040] 5.2 Binding Transfer Experimental Methods Donor and recipient bacteria were cultured separately in LB medium to the logarithmic growth phase. After centrifugation (8000×g, 5 min), the bacterial cells were collected, washed with sterile phosphate buffer (pH 7.2), and resuspended at OD. 600 = 0.4. Mix the two bacterial suspensions at a 1:1 volume ratio and dispense them into sterile EP tubes.

[0041] The experimental setup is as follows for each group: Each treatment group was incubated statically at 30°C for 10 hours. After conjugation transfer, an appropriate amount of bacterial culture was spread onto selective plates containing the corresponding antibiotics and incubated at 30°C for 24 hours before counting the number of transconjugants. The conjugation transfer frequency was calculated using the following formula: Conjugation transfer frequency = number of transconjugants / number of recipient bacteria.

[0042] 5.3 Experimental Results The experimental results are shown in Table 1 and Figure 4 As shown.

[0043] Table 1. Effects of astaxanthin and baicalin on RP4 plasmid binding and transfer frequency. The data listed in the table are the mean ± standard deviation (Mean ± SD) of three independent replicates. Student's t-test was used for significance analysis. Compared with the control group, the binding transfer frequencies of the astaxanthin 1 μg / L and 10 μg / L treatment groups were significantly different (**p<0.01); compared with the baicalin 12.5 μg / mL group, the inhibitory effect of the astaxanthin 1 μg / L group was significantly better than that of the baicalin group (**p<0.01).

[0044] 5.4 Results of the equal concentration comparison experiment To further verify the comparative effect of astaxanthin and baicalin under strictly identical concentration conditions, the inventors set up a control group with equal concentrations of 10 μg / L for both astaxanthin and baicalin. The results showed that, under the same concentration condition of 10 μg / L, astaxanthin exhibited an inhibition rate of 90.2%, while baicalin's inhibition rate was only 43.7%, demonstrating that astaxanthin's inhibitory effect was significantly superior to that of baicalin (p<0.01).

[0045] 5.5 Conclusions of the Comparative Experiment In summary, astaxanthin achieved an inhibition rate of 96.3% at 1 μg / L (four orders of magnitude lower than 12.5 μg / mL of baicalin), which is superior to the 55.8% of baicalin at 12.5 μg / mL. At the same concentration of 10 μg / L, the inhibition rate of astaxanthin (90.2%) was significantly better than that of baicalin at the same concentration (43.7%).

[0046] Example 6 Comparative experiment on the effects of astaxanthin and baicalin on ROS scavenging in the conjugation transfer system 6.1 Experimental Methods Based on the plasmid conjugation transfer system constructed in Example 5, 10 μg / L of antibiotics were added to all treatment groups to promote ROS production. Control group, high-concentration baicalin group (12.5 μg / mL), equal-concentration baicalin group (10 μg / L), low-concentration astaxanthin group (1 μg / L), equal-concentration astaxanthin group (10 μg / L), and high-concentration astaxanthin group (20 μg / L) were set up. The ROS content in each treatment group was determined using the DCFH-DA fluorescent probe method. The mixed bacterial cells of the conjugation transfer system were washed with PBS buffer and resuspended, then DCFH-DA working solution (final concentration 10 μmol / L) was added and incubated at 37°C in the dark for 30 minutes. After washing away probes that did not enter the cells, the fluorescence intensity was measured using a fluorescent microplate reader (Ex / Em = 488 / 525 nm).

[0047] 6.2 Experimental Results The experimental results are shown in Table 2 and Figure 6 As shown.

[0048] Table 2. Scavenging effects of astaxanthin and baicalin on ROS in the RP4 conjugation transfer system. The data listed in the table are the mean ± standard deviation (Mean ± SD) of three independent replicate experiments. Student's t-test was used for significance analysis. Compared with the control group, the ROS levels in each astaxanthin treatment group were significantly different (**p<0.01); compared with the baicalin 12.5 μg / mL group, the ROS scavenging rate of the astaxanthin 1 μg / L group was significantly better than that of the baicalin group (**p*<0.01). The experimental results showed that astaxanthin at 1 μg / L had a significantly higher ROS scavenging rate (66.4%) than baicalin at 12.5 μg / mL (37.9%) (p<0.01); at the same concentration of 10 μg / L, the ROS scavenging rate of astaxanthin (50.9%) was significantly higher than that of baicalin at the same concentration (22.8%) (p<0.01).

[0049] Example 7 Mechanism verification of astaxanthin's inhibition of RP4 plasmid conjugation and transfer 7.1 Observation using laser confocal microscopy The conjugation transfer system was constructed according to the method in Example 5, with a control group (no additives), an antibiotic stress group, and astaxanthin treatment groups (1 μg / L, 20 μg / L). After conjugation transfer, bacterial culture was collected for observation under a laser confocal microscope. The donor bacteria exhibited red fluorescence due to carrying the mCherry gene on their chromosomes; when the RP4 plasmid was transferred into the recipient bacteria that did not carry the lacI^q^ gene via conjugation transfer, the gfp gene on the plasmid was activated, and the transconjugant exhibited green fluorescence.

[0050] Laser confocal observation results as follows Figure 5 As shown in the figure. The results showed that a large number of green fluorescent transconjugants were observed in the antibiotic stress group; the number of green fluorescent transconjugants in the astaxanthin treatment group was significantly reduced (by about 90% compared with the antibiotic stress group), returning to the level of the control group, further confirming the inhibitory effect of astaxanthin on RP4 plasmid conjugation and transfer at the single-cell level.

[0051] 7.2 Analysis of conjugation transfer-related gene expression Total RNA was extracted from donor bacteria in both the antibiotic group and the astaxanthin-treated group (1 μg / L), and the expression levels of conjugation transfer-related genes (trbBp, traF, etc.) were detected by RT-qPCR. The results showed that after astaxanthin treatment, the expression level of the trbBp gene in the donor bacteria decreased by 58.0% ± 1.2% (Ct value increased from 16.3 ± 0.1 to 17.2 ± 0.5) and the expression level of the traF gene decreased by 71.4% ± 1.3% (Ct value increased from 15.7 ± 0.3 to 17.4 ± 0.3), both showing highly significant differences (**p*<0.01). These results indicate that astaxanthin reduces the conjugation transfer ability of the RP4 plasmid by inhibiting the transcriptional expression of conjugation transfer-related genes.

[0052] Example 8 Inhibitory effect of astaxanthin on the spread of ARGs in a bacterial-algae symbiotic wastewater treatment system A symbiotic sludge treatment system based on microorganisms and algae was constructed using the method described in Example 1. Activated sludge was inoculated into an activated sludge reactor, and Haematococcus pluvialis was added to form a stable symbiotic system between the microalgae and the activated sludge. The reactor operating parameters were the same as in Example 1.

[0053] A control group (traditional activated sludge system R0, without microalgae) and an experimental group (bacterial-algae symbiotic system R1) were set up. Antibiotics (tetracycline, kanamycin sulfate, and ampicillin, 500 μg / L each) were added to the influent of both groups. After stable operation, effluent and sludge samples were collected from each reactor, total DNA was extracted, and the abundance of ARGs and MGEs was detected by metagenomic sequencing.

[0054] Metagenomic analysis results as follows Figure 2 As shown in the figure. The results indicate that, compared with the conventional reactor R0, the abundance of ARGs and MGEs in the algal-bacterial symbiotic system R1 was significantly reduced by 77.7% and 77.9%, respectively.

[0055] The results of metabolomics analysis are as follows Figure 3 As shown in the figure. The results indicated that the total astaxanthin content (including intracellular and bound forms) in the algae-bacterial symbiotic system was significantly higher than that of R0 at the same stage, with a difference of 5 to 7 times. In addition, auxiliary antioxidant metabolites such as erythritol, 3-hydroxybutyric acid, yakuchinone A, parthenolide, and russelliside B were also significantly enriched.

[0056] Astaxanthin (and its cofactors) continuously released by Haematococcus pluvialis in a bacterial-algal symbiotic system can effectively reduce the abundance of ARGs and MGEs in wastewater treatment systems and inhibit the spread of ARGs.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of astaxanthin in the preparation of inhibitors, said inhibitors being used to inhibit antibiotic resistance gene conjugation transfer mediated by RP4 plasmid.

2. The application according to claim 1, characterized in that, The concentration of astaxanthin in the inhibitor is 1–20 μg / L.

3. The application according to claim 1, characterized in that, The inhibitor suppresses antibiotic resistance gene conjugation transfer mediated by the RP4 plasmid by reducing intracellular reactive oxygen species levels in bacteria and downregulating the expression of conjugation transfer-related genes.

4. The application according to claim 2, characterized in that, The concentration of astaxanthin is 1–10 μg / L.

5. The application according to any one of claims 1 to 4, characterized in that, The inhibitor is used in wastewater treatment systems to suppress the spread of antibiotic resistance genes.

6. The application according to claim 5, characterized in that, The astaxanthin is produced in situ by Haematococcus pluvialis in a symbiotic sludge treatment system.

7. The application according to claim 6, characterized in that, In the aforementioned algae-bacteria coexisting sludge treatment system, the algae-sludge ratio based on dry weight of Haematococcus pluvialis to activated sludge is 1:2 to 1:8 (w / w), the light intensity is 1000 to 5000 lux, and the dissolved oxygen is maintained at 2 to 4 mg / L.

8. A method for inhibiting antibiotic resistance gene conjugation transfer mediated by RP4 plasmid, characterized in that, include: Add astaxanthin to an environment containing the target microorganism.

9. A bacterial-algae symbiotic sludge treatment system for inhibiting the spread of antibiotic resistance genes, characterized in that, It includes an activated sludge reactor and Haematococcus pluvialis added therein; the ratio of Haematococcus pluvialis to activated sludge based on dry weight is 1:2 to 1:8 (w / w), the light intensity is 1000 to 5000 lux, and the dissolved oxygen is maintained at 2 to 4 mg / L.

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