Method for killing drug-resistant bacteria and blocking drug-resistant gene transmission through photocatalysis

Abstract

The invention discloses a method for killing drug-resistant bacteria and blocking drug-resistant gene transmission through photocatalysis, and belongs to the technical field of environmental functional materials and biological safety. According to the method, a high-entropy spinel oxide composed of six metal elements of Cu, Zn, Ni, Co, Fe and Al is adopted as a photocatalyst. Firstly, a catalyst and a water body containing drug-resistant bacteria are mixed and adsorbed under the dark condition, then visible light is started for a catalytic reaction, generated active oxygen species are utilized for killing the drug-resistant bacteria in situ and degrading extracellular drug-resistant genes (eARGs) released by the drug-resistant bacteria, and finally rapid magnetic separation and recovery are achieved by means of magnetism of the catalyst. According to the method, efficient inactivation (gt; 7-log removal rate) of high-concentration drug-resistant bacteria and synchronous removal of extracellular drug-resistant genes are realized, an adsorption-catalysis-separation integrated process is formed, and the method has the advantages of high efficiency, thoroughness and recyclability and is suitable for deep purification of antibiotic drug-resistant bacteria polluted water.

Description

Technical Field

[0001] This invention relates to the fields of environmental functional materials and biosafety technology, specifically to a method that uses high-entropy spinel oxide as a photocatalyst to efficiently kill multidrug-resistant bacteria carrying broad-host resistance plasmids under visible light, and simultaneously degrades and removes extracellular resistance genes (eARGs) to block their mediated horizontal gene transfer. This method is suitable for the deep purification of water bodies contaminated with high concentrations of drug-resistant bacteria. Background Technology

[0002] The widespread use of antibiotics has led to the proliferation and spread of antibiotic resistance genes (ARGs) in the aquatic environment, posing a global public health and environmental safety challenge. Broad-host-range plasmids (such as RP4) are key vectors that can efficiently spread multiple resistance genes (e.g., resistance to ampicillin, kanamycin, and tetracycline) between different bacterial species via conjugative transfer, accelerating the spread of resistance. Once released into the aquatic environment, RP4 plasmids are readily taken up by environmental microorganisms, leading to a "cascade amplification" effect of resistance.

[0003] Currently, commonly used photocatalytic materials (such as TiO2) can generate reactive oxygen species (ROS) to achieve a certain degree of sterilization, but when treating high concentrations (such as 10⁻⁶), they are less effective. 8 When dealing with multidrug-resistant bacteria (CFU / mL) carrying complex plasmids, there are limitations such as low visible light utilization efficiency, slow sterilization speed, and insufficient ability to remove extracellular resistance genes (eARGs). Although traditional chemical disinfection techniques can inactivate bacteria, they often lead to bacterial lysis and the release of intracellular ARGs into the water, forming an "extracellular gene pool" with a continuous risk of transmission, and there is a lack of effective means to simultaneously remove them.

[0004] Recent studies have shown that hexa-membered high-entropy spinel oxides composed of six metallic elements (Cu, Zn, Ni, Co, Fe, and Al) exhibit good catalytic oxidation performance for small-molecule inorganic pollutants (such as ammonia nitrogen) under visible light. However, the specific adsorption capacity of this material for bacterial cells and their macromolecular genetic material (such as the approximately 60 kb RP4 plasmid), its photocatalytic destruction efficiency, and its potential in blocking horizontal gene transfer have not yet been systematically studied or reported in terms of application. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a method for photocatalytically killing drug-resistant bacteria and blocking the spread of drug-resistant genes. This method can rapidly and thoroughly kill multidrug-resistant bacteria carrying the RP4 plasmid under visible light, and simultaneously degrade and remove the drug-resistant plasmid and its carried gene fragments, thereby blocking the horizontal transfer of drug-resistant genes in the aquatic environment from the source. This is a green and efficient treatment method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] A method for photocatalytic killing of drug-resistant bacteria and blocking the spread of drug-resistant genes includes the following steps:

[0008] (1) Constructing a photocatalytic reaction system: The high-entropy spinel oxide photocatalyst is added to a polluted water body containing Gram-negative bacteria (such as Escherichia coli MG1655 / RP4) carrying the broad host range drug resistance plasmid RP4; the high-entropy spinel oxide is composed of six metal elements Cu, Zn, Ni, Co, Fe and Al in a near equimolar ratio with oxygen element, and has a spinel-type crystal structure.

[0009] (2) Adsorption and photocatalytic degradation:

[0010] Adsorption stage: The mixture is stirred under light-free (dark) conditions to allow the photocatalyst to come into full contact with drug-resistant bacterial cells and free extracellular plasmid DNA in the water. Utilizing the abundant oxygen vacancies and metal active sites on the material surface, the photocatalyst comes into full contact with and adsorbs the drug-resistant bacteria in the water, forming a catalyst-bacteria complex system. This provides a close reaction interface for subsequent light sterilization, achieving adsorption equilibrium.

[0011] Catalytic stage: Irradiation with a visible light source. Under photoexcitation, the catalyst generates photogenerated electron-hole pairs, which are then converted into various highly oxidizing reactive oxygen species (ROS). These ROS attack the bacterial cell walls / membranes adsorbed on the catalyst surface in situ, leading to cell structure destruction, leakage of contents, and death. At the same time, ROS oxidize and degrade extracellular resistance genes (eARGs) released by drug-resistant bacteria, destroying their structural integrity and causing the resistance genes to lose their integrity and biological activity.

[0012] (3) Magnetic separation and recycling: After the photocatalytic reaction, an external magnetic field is applied to the outer wall of the reaction vessel using the inherent magnetic properties of the high-entropy spinel oxide (e.g., saturation magnetization of approximately 1.23 emu / g). The catalyst particles, along with bacterial residues, cell fragments, and degraded gene fragments adsorbed on their surface, rapidly aggregate, achieving rapid (second-level) solid-liquid separation from the treated water. The separated catalyst can be recycled after simple washing and drying, while simultaneously achieving the synergistic removal of organic residues in the water and reducing the bioload of the effluent.

[0013] Furthermore, the preparation method of the high-entropy spinel oxide photocatalyst is as follows: a layered bimetallic hydroxide (LDH) precursor containing the above six metal elements is synthesized by co-precipitation, and then calcined at 500°C in an air atmosphere to obtain the target spinel oxide through topological transformation.

[0014] Furthermore, the specific conditions for the photocatalytic treatment are: a catalyst concentration of 1000 μg / mL and a visible light irradiation time of 30 minutes. Under these optimized conditions, for an initial concentration of approximately 10... 8 The engineered Escherichia coli MG1655 / RP4 strain with CFU / mL and resistance to triple antibiotics (ampicillin, kanamycin, and tetracycline) achieved a kill rate of over 99.9999% (i.e., a removal rate of >7-log), and exhibited significant degradation and removal effects on key resistance genes (bla, tetA, and aphA) on the RP4 plasmid.

[0015] The beneficial effects of this invention are:

[0016] (1) Ultra-efficient killing of multidrug-resistant bacteria: The high-entropy spinel oxide of this invention exhibits extremely strong specific adsorption capacity for drug-resistant bacteria due to its severe lattice distortion and abundant oxygen vacancies on its surface. This synergistic mechanism of "adsorption first, catalysis later" allows photoactive oxygen species to directly and efficiently attack the firmly adsorbed bacteria, thereby achieving ultra-efficient killing of multidrug-resistant bacteria up to 10 8 When treating multidrug-resistant bacteria at CFU / mL, only 30 minutes of visible light irradiation is needed to achieve a kill rate of more than 7 orders of magnitude (>7-log), which is far superior to traditional photocatalysts (such as TiO2) in terms of speed and thoroughness.

[0017] (2) Effectively blocking the horizontal transfer of drug resistance genes: The surface of this material has a strong affinity for plasmid DNA with a negatively charged phosphate backbone, enabling adsorption and enrichment. During photocatalysis, the generated ROS can effectively cleave large molecular plasmid DNA (such as 60kb RP4), destroying its supercoiled structure and the integrity of key drug resistance genes (bla, tetA, aphA). Combined with the material's own magnetism, through magnetic separation after the reaction, not only can the catalyst be recovered, but the adsorbed DNA degradation products can also be physically removed from the water, significantly reducing the abundance and spread risk of eARGs.

[0018] (3) Integrated treatment and material recycling: This invention organically combines the three steps of "adsorption enrichment", "photocatalytic oxidation" and "magnetic separation" to form an integrated deep treatment process of "sterilization-degradation-separation". The catalyst has good magnetic properties, which can be quickly recovered and recycled at least 6 times (the sterilization rate is still >99% after recycling). The recycling process removes organic residues in the water at the same time, reducing the risk of secondary pollution and treatment costs. Attached Figure Description

[0019] Figure 1 These are X-ray diffraction (XRD) patterns of high-entropy spinel oxides (HEO) prepared at different calcination temperatures according to Example 1 of this invention.

[0020] Figure 2This is a comparison of the time-kill curves of HEO-500, TiO2, and blank control MG1655 / RP4 under visible light.

[0021] Figure 3 This is a comparison chart of the bactericidal performance of HEO-500 and commercial TiO2 against MG1655 / RP4 under visible light in Embodiment 3 of the present invention.

[0022] Figure 4 This is a graph showing the change in the relative abundance of three extracellular drug resistance genes (eARGs) in water over time during HEO photocatalytic treatment in Example 4 of this invention.

[0023] Figure 5 This is a schematic diagram illustrating the rapid magnetic separation and recovery achieved by applying an external magnetic field after the HEO-500 photocatalytic reaction.

[0024] Figure 6 These are the results of the cycle stability test of HEO in the photocatalytic killing of Escherichia coli MG1655 / RP4 under visible light.

[0025] Figure 7 This is a schematic diagram of the signal spectrum and reaction mechanism of active oxygen species (•O2⁻, •OH, ¹O2) in the photocatalytic system detected by EPR technology in Example 6 of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Example 1: Preparation of high-entropy spinel oxide (HEO) photocatalyst

[0028] (1) Accurately weigh equimolar amounts (all 0.01 mol) of copper nitrate (Cu(NO3)2·3H2O), zinc nitrate (Zn(NO3)2·6H2O), nickel nitrate (Ni(NO3)2·6H2O), cobalt nitrate (Co(NO3)2·6H2O), iron nitrate (Fe(NO3)3·9H2O) and aluminum nitrate (Al(NO3)3·9H2O), dissolve them together in 50 mL of deionized water, and stir magnetically until completely dissolved to obtain a homogeneous mixed metal salt solution.

[0029] (2) Weigh sodium hydroxide (NaOH) and sodium carbonate (Na2CO3) in a molar ratio of 10:1, dissolve them in an appropriate amount of deionized water, and prepare an alkaline precipitant solution.

[0030] (3) Under continuous stirring, the alkaline precipitant solution prepared in step (2) was slowly added dropwise to the mixed salt solution in step (1). The pH value of the reaction system was stabilized between 9 and 10 by controlling the dropping rate. The reaction was continuously stirred at 25°C for 6 hours. After the reaction was completed, the mixture was allowed to stand for 24 hours. Subsequently, the precipitate was collected by centrifugation and washed repeatedly with deionized water and anhydrous ethanol until the washings were neutral. Finally, the precipitate was dried overnight in a 60°C oven to obtain the high-entropy hydrotalcite (LDH) precursor.

[0031] (4) Divide the dried precursor powder into several portions and place them in porcelain boats in a muffle furnace. In an air atmosphere, heat the powder to 300℃, 500℃, 700℃ and 900℃ respectively at a heating rate of 10℃ / min, and calcine at the target temperature for 4 hours. After calcination, turn off the muffle furnace and allow it to cool naturally to room temperature. After grinding, the powders are obtained as hexa-component high-entropy spinel oxide powders at different calcination temperatures, labeled as HEO-300, HEO-500, HEO-700 and HEO-900 respectively.

[0032] Phase analysis of the sample was performed using X-ray diffraction (XRD), and the results are as follows: Figure 1 As shown, all samples exhibited a single spinel structure characteristic diffraction peak, confirming the successful synthesis of the target product. As the calcination temperature increased from 300℃ to 900℃, the diffraction peak intensity gradually increased, and the full width at half maximum (FWHM) gradually narrowed, indicating that the crystallinity of the material increased with increasing temperature. Considering the balance between catalytic activity and surface defects (such as oxygen vacancies), subsequent experiments verified that the sample calcined at 500℃ (HEO-500) maintained a good crystal structure while possessing the most abundant surface active sites, exhibiting the best photocatalytic bactericidal performance. Therefore, HEO-500 is preferred as the photocatalyst in subsequent application examples.

[0033] Example 2: Construction of a multidrug-resistant engineered bacterial model carrying the RP4 plasmid

[0034] (1) Recipient bacteria and plasmids: Escherichia coli K-12-derived strain MG1655 was selected as the recipient bacteria. This strain has a clear genetic background and does not contain exogenous drug resistance genes. The broad host range conjugation plasmid RP4 was selected as the drug resistance plasmid model. This plasmid carries the ampicillin resistance gene (bla), the kanamycin resistance gene (aphA), and the tetracycline resistance gene (tetA).

[0035] (2) Conjugation transfer and screening: The RP4 plasmid was transferred from the donor bacteria to the recipient bacteria MG1655 by conjugation transfer experiment. The conjugated bacterial culture was spread on LB screening plates containing triple antibiotics (ampicillin Amp 100 μg / mL, kanamycin Km 50 μg / mL, tetracycline Tc 20 μg / mL) and incubated upside down at 37℃ for 16-24 hours.

[0036] (3) Validation and culture of engineered bacteria: Single colonies were picked from the screening plates for expansion culture, and their drug resistance stability was verified by streaking them again on plates containing the same antibiotic. Finally, a multidrug-resistant engineered bacterium carrying the RP4 plasmid was obtained and named MG1655 / RP4. The engineered bacteria were inoculated into LB liquid medium (without antibiotics) and cultured at 37°C and 200 rpm with shaking until the mid-logarithmic growth phase (OD600≈0.6). The bacterial cells were collected by centrifugation, washed twice with sterile physiological saline to remove the culture medium components, and finally resuspended in sterile physiological saline.

[0037] (4) Preparation of bacterial suspension: The concentration of the bacterial suspension was accurately determined by plate counting method, and diluted with sterile physiological saline to a concentration of approximately 1×10⁻⁶. 8 The concentration of CFU / mL was used to simulate high concentrations of antibiotic-resistant bacteria contaminating water bodies for subsequent photocatalysis experiments.

[0038] Example 3: Evaluation of rapid inactivation efficacy against multidrug-resistant bacteria MG1655 / RP4 under visible light

[0039] (1) Experimental setup: Accurately weigh 50.0 mg of the HEO-500 catalyst prepared in Example 1 and add it to 50 mL of the MG1655 / RP4 bacterial suspension prepared in Example 2 (10 8 In a concentration of CFU / mL, the catalyst was brought to a final concentration of 1000 μg / mL. Two control groups were set up: ① Blank control group (bacterial suspension only, no catalyst, same light); ② Traditional material control group (50.0 mg TiO2 was used instead of HEO-500, other conditions were the same).

[0040] (2) Adsorption equilibrium: Place the above mixture on a magnetic stirrer and stir continuously for 30 minutes at room temperature and in complete darkness to allow the catalyst to come into full contact with the bacteria and reach adsorption equilibrium.

[0041] (3) Photocatalytic reaction: Turn on the 500 W xenon lamp light source (install a 420 nm cutoff filter in front of the light source to filter out ultraviolet light and provide pure visible light irradiation), start the timer and keep stirring. Take samples at 0, 10, 20 and 30 minutes of illumination.

[0042] (4) Bacterial viability determination: After each sampling, appropriate tenfold serial dilutions were performed immediately. 100 μL of the bacterial solution at a specific dilution was spread on an antibiotic-free LB agar plate, with three replicates for each time point. All plates were incubated upside down in a 37°C incubator for 16-24 hours, followed by colony counting and calculation of the viable bacterial concentration (CFU / mL).

[0043] (5) Results Analysis:

[0044] HEO-500 treatment group: Results are as follows Figure 2 and Figure 3 As shown, under visible light irradiation, the survival concentration of MG1655 / RP4 decreased exponentially over time. After 30 minutes of irradiation, no colonies grew on the spread plates, indicating that the bacterial concentration decreased from the initial ~10⁻⁶. 8 When the CFU / mL was reduced to below the method detection limit (<10 CFU / mL), the kill rate exceeded 99.999999%, achieving a removal of 8 orders of magnitude (8-log).

[0045] TiO2 control group: as shown Figure 2 The comparison shows that, under the same treatment conditions, TiO2 has extremely limited effectiveness in killing high concentrations of drug-resistant bacteria. After 30 minutes of irradiation, the bacterial concentration only decreased from 10... 8 CFU / mL decreased to 10 7 The amount removed is approximately CFU / mL, which is less than one order of magnitude (1-log).

[0046] (6) Conclusion: The high-entropy spinel oxide HEO-500 of the present invention, through the strong adsorption mediated by oxygen vacancies on its surface, efficiently enriches bacteria near the active site of the catalyst, realizing the in-situ and efficient oxidation attack of bacteria by photoactive oxygen species, forming a powerful "adsorption-catalysis" synergistic bactericidal effect, and its treatment efficiency is significantly better than that of traditional semiconductor photocatalytic materials.

[0047] Example 4: Validation of the synergistic clearance effect of RP4 plasmid and extracellular drug resistance genes (eARGs)

[0048] (1) Sample collection: In the HEO-500 treatment experiment of Example 3, water samples were collected at 0 minutes, 10 minutes, 20 minutes and 30 minutes of light exposure. At the same time, a blank control group (bacterial suspension only, light exposure for 30 minutes) was set up.

[0049] (2) DNA extraction: Use an environmental genomic DNA extraction kit suitable for water samples and strictly follow the instructions to extract total DNA (including free extracellular DNA and DNA released by bacterial lysis) from each water sample.

[0050] (3) qPCR quantitative analysis: Specific primers were designed for the three characteristic drug resistance genes on the RP4 plasmid: bla (ampicillin resistance), tetA (tetracycline resistance), and aphA (kanamycin resistance). Absolute quantification was performed using real-time quantitative PCR (qPCR). A standard curve was constructed using plasmid standards containing the target gene fragments, and the absolute copy number concentrations of the three genes in each water sample were calculated.

[0051] (4) Data processing: The copy number of each drug resistance gene in the HEO treatment group at 0 minutes was used as the initial reference value (set to 1) to characterize the relative change trend of extracellular drug resistance genes over time during HEO photocatalytic treatment. At the same time, the same normalization method was used to process the light-illuminated blank control group to compare the change behavior of drug resistance gene abundance in different treatment systems under light conditions.

[0052] (5) Results and Discussion: The results are as follows Figure 4 As shown.

[0053] In the light-controlled blank control group, the relative abundance of the three drug resistance genes did not change significantly throughout the experiment and remained basically at the initial level.

[0054] In the HEO-500 visible light treatment group, the abundance of the three extracellular resistance genes (eARGs) showed a continuous and significant decreasing trend as the photocatalytic reaction proceeded. After 30 minutes of illumination, the relative abundance of bla, tetA, and aphA genes decreased by approximately 78%, 75%, and 80%, respectively, equivalent to a reduction of 0.6–0.8 log.

[0055] (6) Conclusion: The method of this invention, while killing bacteria, can effectively degrade extracellular resistance genes (eARGs) released by drug-resistant bacteria through photocatalysis, thereby disrupting their genetic integrity. More importantly, combined with the magnetism of the HEO material, the DNA degradation products adsorbed on the catalyst surface can be physically removed from the water body through magnetic separation after the reaction. This dual removal mechanism, combining "chemical oxidation degradation" and "physical adsorption separation," can significantly reduce the level of genetic material with potential biological activity and horizontal transfer risk in water bodies, thereby effectively controlling the environmental spread of drug resistance genes.

[0056] Example 5: Stability Test of Magnetic Recovery and Recycling of Photocatalyst

[0057] (1) Demonstration of magnetic separation: After the reaction in Example 3 was completed (30 minutes of light exposure), a strong neodymium iron boron magnet was placed on the outer wall of the glass beaker containing the reaction solution. It can be observed that the suspension quickly became clear within 1 minute, and the black HEO-500 powder (whose surface had been adsorbed with bacterial remains, cell debris and degradation products) was attracted by the magnetic force, quickly agglomerated and firmly attached to the inner wall of the container near the magnet. Figure 5 Pour out the supernatant to complete the solid-liquid separation.

[0058] (2) Catalyst recovery and regeneration: The accumulated catalyst was scraped off the container wall and transferred to a centrifuge tube. It was ultrasonically washed three times each with deionized water and anhydrous ethanol for 5 minutes each time to remove the organic residues adsorbed on the surface. After centrifugation, the supernatant was discarded, and the catalyst solid was dried in an oven at 60°C for 6 hours to obtain the regenerated HEO-500 catalyst.

[0059] (3) Cyclic performance test: The regenerated catalyst was used again to kill MG1655 / RP4 under the same experimental conditions as in Example 3 (catalyst dosage 1000 μg / mL, light irradiation for 30 minutes). After completing one sterilization experiment, magnetic separation and washing regeneration were performed again according to steps (1) and (2). The cycle of "reaction-magnetic separation-washing-re-reaction" was repeated.

[0060] (4) Results analysis: such as Figure 6 As shown, the sterilization efficiency of the HEO-500 catalyst was recorded after six consecutive cycles. The results indicate that even after six cycles, the catalyst maintained a sterilization rate of over 99% against MG1655 / RP4 under 30 minutes of visible light irradiation.

[0061] (5) Conclusion: The high-entropy spinel oxide HEO-500 prepared in this invention has good magnetic properties (saturation magnetization of about 1.23 emu / g), enabling rapid magnetic separation and efficient recovery. Its crystal structure is stable and it can maintain excellent photocatalytic activity after multiple cycles. At the same time, the magnetic separation process not only recovers the catalyst, but also synergistically removes biological debris and organic degradation products attached to its surface, further reducing the biological load of the effluent and achieving the dual purpose of catalyst recycling and water purification.

[0062] Example 6: Investigation of the photocatalytic reaction mechanism

[0063] To further elucidate the microscopic mechanism of the high-entropy spinel oxide (HEO) of this invention in achieving efficient sterilization and gene degradation under visible light, electron paramagnetic resonance (EPR) technology was used to directly capture and identify reactive oxygen species (ROS) generated during photocatalysis.

[0064] (1) Experimental method:

[0065] Prepare an aqueous dispersion of HEO-500.

[0066] Different spin trapping agents were used: 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) was used to trap superoxide radicals (•O2⁻) and hydroxyl radicals (•OH); 2,2,6,6-tetramethylpiperidine (TEMP) was used to trap singlet oxygen (¹O2).

[0067] The HEO dispersion was mixed with either DMPO or TEMP scavenging agent and divided into two portions. One portion was placed in the dark, while the other portion was irradiated with visible light (500 W xenon lamp with a 420 nm cutoff filter) for 5 minutes.

[0068] Immediately perform EPR spectroscopy on the sample.

[0069] (2) EPR results: such as Figure 7 As shown in (ac).

[0070] Figure 7 (a): No signal under dark conditions; after visible light irradiation, a quartet with an intensity ratio of 1:1:1:1 appears, which is the characteristic signal of the DMPO-•O2⁻ adduct, proving that •O2⁻ is generated in the system.

[0071] Figure 7 (b): After visible light irradiation, a quartet with an intensity ratio of 1:2:2:1 appears, which is a characteristic signal of the DMPO-•OH adduct, proving that •OH is generated. This may be indirectly generated through pathways such as the disproportionation of •O2⁻.

[0072] Figure 7 (c): After irradiation with visible light, a triplet with an intensity ratio of 1:1:1 appears. This is a characteristic signal of the TEMP-¹O2 adduct, proving that ¹O2 is generated.

[0073] (3) Mechanism explanation: Combining EPR results and band structure analysis of the material (see schematic diagram) Figure 7The photocatalytic mechanism of the high-entropy spinel oxide of this invention can be described as follows: Under visible light excitation, HEO generates photogenerated electrons (e⁻) and holes (h⁺). The photogenerated electrons reduce adsorbed O₂ on the surface to •O₂⁻, which can further generate secondary reactive species such as ¹O₂ and •OH through conversion reactions; simultaneously, the photogenerated holes also possess strong oxidizing capabilities. These generated reactive oxygen species (mainly including ¹O₂, •OH, and •O₂⁻) work synergistically to launch a highly efficient attack on the cell walls / membranes of drug-resistant bacteria strongly adsorbed on the HEO surface, leading to rapid cell death; simultaneously, these strong oxidizing species can also oxidize and cleave adsorbed plasmid DNA nearby, disrupting its structural integrity, thereby achieving simultaneous "bacterial killing" and "gene degradation".

[0074] The above embodiments fully verify the effectiveness, efficiency, and sustainability of the high-entropy spinel oxide provided by the present invention in the application of photocatalytic killing of antibiotic-resistant bacteria and blocking the spread of drug-resistant genes.

Claims

1. A method for photocatalytically killing drug-resistant bacteria and blocking the spread of drug-resistant genes, characterized in that, Includes the following steps: (1) A photocatalytic reaction system was constructed by adding high-entropy spinel oxide photocatalyst to water bodies contaminated with drug-resistant bacteria carrying broad-host-range drug-resistant plasmids; (2) Stirring under light-free conditions to allow the photocatalyst to come into full contact with and be adsorbed by drug-resistant bacteria in the water, forming a catalyst-bacteria composite system; (3) Turn on the visible light source to carry out photocatalytic reaction, and generate active oxygen species through photocatalysis to kill the adsorbed drug-resistant bacteria and degrade the extracellular drug resistance genes eARGs released by the drug-resistant bacteria, thereby blocking the environmental spread of drug resistance genes. (4) After the reaction is completed, the photocatalyst is magnetically separated and recovered using an external magnetic field to achieve solid-liquid separation.

2. The method according to claim 1, characterized in that, The high-entropy spinel oxide is composed of six metal elements, Cu, Zn, Ni, Co, Fe, and Al, and oxygen, with the molar ratio of the six metal elements being nearly equal, and has a spinel-type crystal structure.

3. The method according to claim 2, characterized in that, The high-entropy spinel oxide is prepared by: synthesizing a layered bimetallic hydroxide precursor containing the six metal elements by co-precipitation, followed by calcination at 500°C in air to obtain it through topological transformation.

4. The method according to claim 1, characterized in that, The concentration of the photocatalyst is 1000 μg / mL, and the irradiation time with visible light is 30 minutes.

5. The method according to claim 1, characterized in that, The drug-resistant plasmid is the RP4 plasmid, and the drug-resistant bacterium is Escherichia coli MG1655 / RP4 carrying the RP4 plasmid.

6. The method according to claim 1 or 5, characterized in that, The reactive oxygen species generated by the photocatalytic reaction include superoxide radical •O2⁻, hydroxyl radical •OH, and singlet oxygen¹O2.

7. A magnetic high-entropy spinel oxide photocatalyst for carrying out the method according to any one of claims 1-6, characterized in that, The catalyst is composed of six metallic elements (Cu, Zn, Ni, Co, Fe, and Al) and oxygen in a near equimolar ratio, has a spinel-type crystal structure, and has a saturation magnetization of 1.23 emu / g.

8. The magnetic high-entropy spinel oxide photocatalyst according to claim 7, characterized in that, It is prepared by calcining a layered bimetallic hydroxide precursor containing the six metal elements at 500°C.

9. A system for integrated purification of drug-resistant bacteria and drug-resistant genes, characterized in that, The system includes: A reaction unit for containing the water to be treated and adding the magnetic high-entropy spinel oxide photocatalyst as described in claim 7 or 8; The light source unit is used to provide visible light irradiation to drive the photocatalytic reaction; The magnetic separation unit is used to achieve rapid separation of the catalyst from the treated water by applying an external magnetic field after the reaction is completed.

10. The system according to claim 9, characterized in that, The system operation process includes sequential adsorption, photocatalytic oxidation, and magnetic separation steps.

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