Method for inhibiting resistant gene conjugational transfer based on Mo monatomic catalyst activated peroxydisulfate system

Abstract

The invention discloses a method for inhibiting resistant gene conjugational transfer based on a Mo monatomic catalyst activated peroxydisulfate system. The method is characterized in that the Mo monatomic catalyst activated peroxydisulfate system inhibits resistance gene conjugational transfer by activating oxidative stress response, destroying membrane permeability, interfering energy metabolism and inhibiting biological membrane formation, the Mo monatomic catalyst is a Mo / NC catalyst, and the catalyst can efficiently activate PDS to generate active species 1O2 and SO4. The method realizes effective removal of ARB and ARGs in water, and active species induce oxidative stress to trigger SOS reaction in the reaction process, so as to inhibit ATP synthesis and reduce energy supply, thereby inhibiting conjugation transfer of ARGs. Scientific basis and technical solution can be provided for promoting treatment of drug-resistant pollution in the water environment.

Description

Technical Field

[0001] This invention belongs to the field of antibiotic-resistant bacteria and resistance gene inhibition technology, specifically relating to a method for inhibiting resistance gene conjugation and transfer based on a Mo single-atom catalyst-activated persulfate system. Background Technology

[0002] Antibiotics, currently the most widely used and diverse class of antibacterial drugs both domestically and internationally, are primarily used to treat bacterial infectious diseases and as animal feed additives in animal husbandry and aquaculture to stimulate animal growth and prevent disease. However, antibiotics entering the body cannot be completely metabolized and absorbed; residual antibiotics are excreted in human and animal feces and urine, entering water bodies and inducing the production of large numbers of antibiotic-resistant bacteria (ARBs) and antibiotic-resistant genes (ARGs). On the one hand, ARGs are vertically transferred to offspring bacteria, leading to an increase in ARBs in the environment, altering the microbial structure and community of the ecosystem, and disrupting ecological balance. On the other hand, ARGs also undergo horizontal gene transfer (HGT) between bacteria of the same or different species through binding, transformation, and transduction, enabling antibiotic-sensitive bacteria to acquire resistance and even leading to the emergence and spread of multidrug-resistant pathogens, increasing the difficulty of treating human diseases. It is estimated that by 2050, the number of people worldwide who will die from ARB infections will reach 10 million. The World Health Organization has listed ARGs as one of the major challenges threatening human health in the 21st century and announced a global strategic deployment for ARG control. Compared with chemical pollutants, ARGs are "persistent" and "replicable," easily re-expressed through repair, making it difficult to fundamentally eliminate the risk of resistance gene transfer. While disinfection technologies such as ultraviolet light, chlorination, and ozone can inactivate some ARBs, they cannot fundamentally reduce resistance genes and eliminate the risk of drug resistance. Therefore, developing safe and efficient key technologies for reducing ARBs and ARGs in water is of great significance for inhibiting the horizontal transfer of resistance genes.

[0003] Advanced oxidation techniques based on peroxydisulfate (PDS) can generate highly oxidizing sulfate radicals (SO4) by activating persulfate with a catalyst. ·- ), hydroxyl radicals (·OH) and singlet oxygen ( 1O2), these active species can directly disrupt bacterial cell membranes, efficiently inactivating ARBs and degrading ARGs, and inhibiting ARG transfer. Furthermore, this disinfection technology has strong oxidizing power, good stability, and is minimally affected by water pH, exhibiting high disinfection capabilities even in complex water bodies, thus possessing wide applicability. Currently, catalysts used to activate PDS mainly include transition metals, carbon materials, and single-atom catalysts. However, transition metal ions suffer from severe leakage, and carbon materials have low activation efficiency. Single-atom catalysts, due to their interaction with the support, allow each active atom to be exposed on the surface to participate in the reaction, maintaining this performance over long-term use and maximizing the utilization of metal catalytic activity. Currently, the most researched are metal-NC single-atom catalysts (M / NC), where M represents a transition metal, C represents a carbon material, and N can form a coordination bond with a single metal atom, serving as the active site for single-atom catalyst activation of PDS. However, the catalytic efficiency of such materials is still limited by the slow proton-coupled electron transfer process. The most effective way to solve this problem is to adjust the electronic microenvironment around the metal site to accelerate catalyst electron transfer. Recent studies have found that by changing the coordination number of N atoms, the microscopic electronic structure near single-atom sites can be modulated, thereby enhancing the activity of M / NC. Therefore, it is feasible to construct asymmetric electronic structures with different N coordinations to alter the electronic microenvironment around active sites, enhance electron transfer between active sites, and ultimately achieve efficient activation of persulfate.

[0004] This invention aims to obtain M / NC single-atom catalysts with different coordination environments by changing the calcination temperature, breaking the symmetry of the Mo-N4 structure, altering the electron density around the Mo sites, and increasing the electron transfer rate between the catalyst and PDS, thereby achieving efficient PDS activation. Based on this, *E. coli* carrying the RP4 plasmid was selected as the target strain to investigate the effects of the M / NC-activated PDS system on ARB inactivation and ARG degradation. By studying the changes in ARB oxidative stress, cell membrane permeability, energy metabolism, and biomembrane in the system, combined with gene sequencing technology, the mechanism of inhibiting ARG conjugation and translocation was revealed. This invention provides guidance for ARB inactivation, ARG degradation, and inhibition of ARG conjugation and translocation, offering new data support and theoretical backing for the protection of water resources and the environment. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a method for inhibiting the conjugation and transfer of resistance genes based on a Mo single-atom catalyst-activated peroxydisulfate (PDS) system. This method constructs a highly efficient Mo / NC single-atom nitrogen-carbon catalyst (Mo / NC) activated peroxydisulfate (PDS) system, and systematically studies the removal efficiency of ARBs and ARGs in water, providing theoretical basis and technical support for water pollution control strategies. A series of Mo / NC catalysts were prepared by secondary calcination using calcium citrate, ammonium molybdate, and ammonium chloride as precursors. The microstructure, crystal structure, elemental composition, chemical valence state, and coordination environment of the Mo / NC catalysts were analyzed using various characterization methods. Using resistant *E. coli* carrying the RP4 plasmid as a model organism, the reduction effect of the Mo / NC catalyst-activated PDS system on ARBs and ARGs was studied. The changes in the antioxidant capacity and cell membrane permeability of ARBs during sterilization were analyzed to further investigate the reasons for ARB inactivation. The activation mechanism of PDS was revealed through active site identification, active species analysis, and electrochemical testing. A conjugation transfer model was constructed to evaluate the inhibitory effect of the Mo / NC catalyst-activated PDS system on ARG conjugation transfer. Using bacterial transcriptome sequencing, the expression changes of key functional genes during conjugation transfer, such as genes related to cell membrane permeability, genes related to the oxidative stress-SOS response pathway, genes related to adenosine triphosphate (ATP) metabolism, and genes regulating biofilm formation, were analyzed. The inhibitory effect of the Mo / NC catalyst-activated PDS system on the horizontal transfer of ARGs was systematically analyzed.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for inhibiting the binding and transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system. The method is characterized in that: the Mo single-atom catalyst-activated persulfate system inhibits the binding and transfer of resistance genes by activating oxidative stress response, disrupting membrane permeability, interfering with energy metabolism, and inhibiting biomembrane formation. The Mo single-atom catalyst is a Mo / NC catalyst, prepared by calcining anhydrous calcium citrate at 700–900°C to form a C material, then mixing the C material with ammonium molybdate and ammonium chloride to form a precursor, which is then calcined at 400–600°C.

[0007] Furthermore, the Mo single-atom catalyst can efficiently activate persulfate to generate active species. 1 O2 and SO4 ·- This process effectively removes antibiotic-resistant bacteria and antibiotic resistance genes from water bodies. During the reaction, the active species induce oxidative stress in antibiotic-resistant bacteria, triggering the SOS response, which enhances the permeability of the antibiotic-resistant bacterial cell membrane, inhibits the ATP synthesis pathway, and reduces energy supply, thereby inhibiting the conjugation and transfer of antibiotic resistance genes.

[0008] Further specifying, the antibiotic resistance genes are the tetA gene, the APH-Ib gene, and the blaTEM-2 gene.

[0009] Further, the application of the Mo single-atom catalyst-activated persulfate system in the preparation of drug-resistant pollution control agents for aquatic environments is defined.

[0010] Further specifying the preparation process of the Mo single-atom catalyst, the following steps are taken: Anhydrous calcium citrate is calcined in a tube furnace at a heating rate of 2–5 °C / min to 700–900 °C for 1–2 h under nitrogen atmosphere. The calcined powder is thoroughly ground and stirred overnight in hydrochloric acid. It is then washed with ultrapure water until the solution is neutral and dried to obtain C material. 0.1924 g of the prepared C material is dispersed in 10 mL of an aqueous solution containing 0.0144 g of ammonium molybdate and 0.5000 g of ammonium chloride. The solution is stirred at 80–90 °C until completely dry, then transferred to a tube furnace and calcined at 400–600 °C at a heating rate of 2–5 °C / min under nitrogen atmosphere for 2–3 h. The solution is then naturally cooled to room temperature to obtain a Mo single-atom catalyst with a Mo mass percentage of 4%.

[0011] Further specifying, the preferred calcination temperature is 400℃.

[0012] This invention offers the following advantages and benefits: It prepares a Mo single-atom catalyst capable of efficiently activating PDS by altering the Mo coordination environment through calcination. The catalytic activity of the Mo / NC catalyst is evaluated using ARBs and ARGs as target pollutants. The inactivation mechanisms of ARBs are analyzed in detail through oxidative stress and cell membrane permeability analysis, and the activation mechanism of PDS is proposed through active site and bioactive species analysis. Finally, an ARGs conjugation and transfer model is constructed to analyze the horizontal transfer frequencies of ARGs under conditions of donor bacteria treatment only, recipient bacteria treatment only, and simultaneous treatment with both donor and recipient bacteria. Transcriptome sequencing reveals the reasons for the system's inhibition of ARGs horizontal transfer. This invention provides fundamental data and technical support for the application of reducing ARBs and ARGs in water bodies. Specific research conclusions are as follows:

[0013] (1) The present invention prepares Mo / NC catalyst by high temperature calcination. In the catalyst, Mo is dispersed on the C-based material in the form of single atoms. The change of calcination temperature causes the change of the microenvironment around the Mo-N site. The disorder of the Mo / NC catalyst provides more active sites for the reaction.

[0014] (2) The Mo / NC-400 catalyst prepared in this invention can inactivate 7.3 log CFU / mL of PDS system within 100 min. -1 The ARB. During sterilization, the Mo / NC-400 catalyst can effectively adsorb PDS, and electron transfer to PDS leads to its activation, generating a large amount of...1 O2 and SO4 ·- These bioactive species continuously attack bacteria, causing oxidative stress and increasing intracellular SOD and CAT concentrations to counteract the attack. When intracellular ROS levels exceed the tolerance of the bacterial defense system, the antioxidant system is damaged, and the concentrations of SOD and CAT decrease, leading to an inability to effectively clear intracellular ROS and ultimately cell death. Compared to carbon materials, the Mo / NC-400 catalyst can inactivate 7.3 log CFU / mL of PDS in a PDS activation system within 100 min. - 1 The number of ARB target genes tetA, APH-Ib, blaTEM-2, and housekeeping gene 16S rRNA decreased by 5.23 log, 4.94 log, 4.26 log, and 3.86 log, respectively. Analysis of bacterial antioxidant capacity and cell membrane permeability showed that the activation of PDS by the Mo / NC-400 catalyst to inactivate ARB triggered oxidative stress in bacteria, increasing cell membrane permeability and leading to bacterial rupture and even death. The main active sites of Mo / NC-400 are Mo-N and pyridine N. Electrons are transferred to the adsorbed PDS through these active sites, leading to PDS activation and the generation of active species. Combining electron paramagnetic resonance (EPR) technology and quenching experiments, it was found that... 1 O2 plays a major role, SO4 ·- It plays a supporting role.

[0015] (3) In this system, the target genes tetA, APH-Ib, blaTEM-2, and the housekeeping gene 16S rRNA were degraded by 5.23 log, 4.94 log, 4.26 log, and 3.86 log, respectively. An ARGs transfer model was constructed to analyze the horizontal transfer frequency of ARGs under three conditions. When only the donor bacteria were treated, conjugation transfer was completely inhibited; when only the recipient bacteria were treated, or when both donor and recipient bacteria were treated simultaneously, the conjugation transfer frequency showed a trend of first increasing and then decreasing. Based on analyses of oxidative stress, cell membrane permeability, ATP synthesis, and transcriptome sequencing, the reason for the inhibition of ARGs conjugation transfer was proposed: oxidative stress was generated during ARB inactivation, cell membrane permeability was damaged, energy metabolism was inhibited, and ATP synthesis was limited, ultimately leading to a decrease in the conjugation transfer frequency. The results of the three conjugation transfer models showed that when only the donor bacteria were treated, the conjugation frequency gradually decreased, and conjugation transfer was completely inhibited at 60 min. When only the recipient bacteria were treated, or when both donor and recipient bacteria were treated simultaneously, the conjugation transfer frequency first increased and then decreased. Transcriptomic analysis revealed that the Mo / NC-400 catalyst-activated PDS system inhibited the horizontal transfer of ARGs through pathways such as activating oxidative stress response, disrupting membrane permeability, interfering with energy metabolism, and inhibiting biomembrane formation.

[0016] This invention constructs a Mo / NC catalyst-activated PDS system, studies its removal efficiency for ARBs and ARGs, and investigates the causes of resistant gene horizontal transfer. The results show that the Mo / NC catalyst can efficiently activate PDS to produce active species. 1 O2 and SO4 ·- This invention achieves effective removal of ARBs and ARGs from water bodies. During the reaction, the active species induce oxidative stress, triggering the SOS reaction, inhibiting ATP synthesis and reducing energy supply, thereby suppressing the binding and transfer of ARGs. This invention can provide a scientific basis and technical solution for promoting the remediation of drug-resistant pollution in aquatic environments. Attached Figure Description

[0017] Figure 1 The standard curves for qPCR of the target genes are as follows: 16S rRNA (a); APH-Ib (b); tetA (c) and blaTEM-2 (d).

[0018] Figure 2 HRTEM and HAADF-STEM images of samples calcined at different temperatures: HRTEM image of Mo / NC-400 (a, b); HRTEM image of Mo / NC-500 (c); HRTEM image of Mo / NC-600 (d); HAADF-STEM image of Mo / NC-400 (e); EDS image of Mo / NC-400 (f).

[0019] Figure 3 For structural analysis of samples calcined at different temperatures, XRD analysis of the samples (a); Raman spectrum of the samples (b); XPS analysis: full spectrum (c); high-resolution spectra of C 1s (d), N 1s (e) and Mo 3d (f).

[0020] Figure 4 To illustrate the sterilization effect, total DNA concentration changes, and target gene degradation of samples at different calcination temperatures, the sterilization effect of individual samples on bacteria is shown in (a); the sterilization effect of the sample-activated PDS system on bacteria is shown in (b); the total DNA concentration changes are shown in (c); and the changes in the quantities of tetA, APH-Ib, blaTEM-2, and 16S rRNA are shown in (d).

[0021] Figure 5 The effects of Mo / NC-400-activated PDS on bacterial oxidative damage: intracellular ROS level (a); SOD and CAT activities (b); LDH content (c).

[0022] Figure 6SEM and TEM analyses of bacteria before and after Mo / NC-400 treatment; SEM analysis of bacteria at 0 min (a) and 100 min (b); TEM analysis of bacteria at 0 min (c) and 100 min (d).

[0023] Figure 7 The degradation of PDS in the Mo / NC-400 activated PDS system, the results of the premixing experiment, and the EPR analysis are shown. PDS degradation rate (a); premixing experiment results (b); ·OH / SO4 ·- (c); O2 ·- (d); 1 O2(e, f).

[0024] Figure 8 Identification of active species in the Mo / NC-400 activated PDS system, quenching experiment (a); PMSO and PMSO2 content (b); effect of N2 on sterilization effect (c).

[0025] Figure 9 For the quantitative detection of free radicals in the Mo / NC-400 activated PDS system, 1 Quantitative detection of O2 (a); quantitative detection of ·OH (b).

[0026] Figure 10 Electrochemical analysis of the Mo / NC-400 activated PDS system: LSV curve (a); it curve (b).

[0027] Figure 11 The sterilization effects of catalysts treated with KSCN, H3PO4 and K2CO3 are shown in the following figures: sterilization effect of catalyst treated with KSCN (a); sterilization effect of catalysts treated with H3PO4 and K2CO3: Mo / NC-400 (b), Mo / NC-500 (c), Mo / NC-600 (d).

[0028] Figure 12 The effect of the Mo / NC-400 activated PDS system on ARGs conjugation transfer: recipient bacteria number (a); conjugate number (b); conjugation transfer frequency (c).

[0029] Figure 13 Effects of Mo / NC-400 treatment on oxidative stress and ATP synthesis in donor and recipient bacteria: intracellular ROS content (a); MDA content (b); ATP content (c).

[0030] Figure 14 Changes in biomass of donor and recipient bacteria biofilms (a); content of protein (b), polysaccharide (c), and eDNA in EPS (d).

[0031] Figure 15 This indicates the oxidative stress and cell membrane-related gene expression status of the donor bacteria.

[0032] Figure 16 The expression of genes related to ATP synthesis in the donor bacteria.

[0033] Figure 17 The expression of genes related to quorum sensing, biomembranes, and EPS. Detailed Implementation

[0034] Example

[0035] 1. Methods and Experiments

[0036] 1.1 Material Preparation

[0037] (1) Take 3g of anhydrous calcium citrate and calcine it in a tube furnace at 800℃ for 1h (N2, 3℃min). -1 The calcined powder was thoroughly ground and stirred overnight in a 10% (v / v) hydrochloric acid solution. It was then washed with ultrapure water until the solution was neutral (pH = 7) and dried in an 80°C oven to obtain material C. The prepared material (0.1924 g) was dispersed in 10 mL of an aqueous solution containing 0.0144 g ammonium molybdate and 0.5000 g ammonium chloride. After stirring at 80°C until completely dry, it was transferred to a tube furnace and calcined at 400°C for 2 h (N2, 3°C min). -1 The catalyst was naturally cooled to room temperature to obtain a Mo single-atom catalyst with a Mo mass percentage of 4%, named Mo / NC-400. CN was prepared without the addition of ammonium molybdate.

[0038] (2) In addition, Mo single-atom catalysts with a Mo mass percentage of 4% were prepared at calcination temperatures of 500℃ and 600℃, respectively, and named Mo / NC-500 and Mo / NC-600.

[0039] 1.2 ARB inactivation experiment and ARGs degradation test

[0040] 1.2.1 ARB inactivation experiment

[0041] Weigh 0.1 g PDS and dissolve it in 4 mL of ultrapure water to prepare a 6 mM stock solution for later use; weigh 0.12 g Mo single-atom catalyst and add it to 3 mL of ultrapure water for ultrasonic dispersion. Take 125 μL of Mo single-atom catalyst suspension and 432 μL of PDS and add it to a solution containing 10 7 CFU mL -1Samples were taken from 10 mL of bacterial solution at 0, 25, 50, 75, and 100 min. Immediately after each sample was taken, 10 mM sodium thiosulfate was added to quench PDS and terminate the reaction. The samples were then serially diluted, plated, and incubated at 37°C for 24 h. Bacterial counts were performed according to GB4789.2-2016. Sterilization experiments with only PDS or Mo / NC served as controls. All ultrapure water and glassware used were autoclaved, and all experiments were repeated three times.

[0042] 1.2.2 ARGs Detection

[0043] (1) DNA extraction

[0044] The total volume of bacterial suspension for the DNA extraction experiment was 600 mL. 100 mL of bacterial suspension was collected from the Mo / NC-400 activated PDS system at time intervals of 0, 25, 50, 75, and 100 min. The precipitate was collected by centrifugation at 3400 × g for 10 min. Following the instructions of the Spin Kit for Soil, DNA was extracted, and the concentration and purity of the sample DNA were determined using a microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific, USA). The obtained DNA sample was stored at -20°C.

[0045] (2) Genetic testing

[0046] ① Preparation of the reaction system: see Table 1.

[0047] Table 1 PCR reaction system

[0048]

[0049] ②PCR cycling programs for APH-Ib, blaTEM-2, and tetA: see Table 2.

[0050] Table 2 PCR Cyclic Program

[0051]

[0052] ③PCR cycle program for 16S rRNA: see Table 3.

[0053] Table 3 PCR Cycling Program

[0054]

[0055] ④ Primer sequences for the four genes: see Table 4.

[0056] Table 4. Gene sequences used in PCR experiments.

[0057]

[0058] ⑤ Agarose gel electrophoresis: Take 10 mL of PCR product and verify it by electrophoresis on a 1.5% agarose gel at 90V and 1×TAE buffer for 40 min.

[0059] ⑥ Exposure: The target fragment was observed using a chemiluminescent gel imaging system, and the PCR product was sequenced by Shanghai Sangon Biotech Co., Ltd. The primers were synthesized by Shanghai Sangon Biotech.

[0060] (3) Real-time quantitative PCR

[0061] ① Preparation of the reaction system: see Table 5.

[0062] Table 5 qPCR reaction system

[0063]

[0064] ② qPCR cycling program: The target gene was quantitatively analyzed using a qPCR instrument (Roche, Switzerland). A two-step temperature control method was used, with the pre-denaturation temperature set at 95℃ to fully activate the hot-start enzyme. The program was as follows: pre-denaturation 94℃, 30s; denaturation 94℃, 5s; annealing / extension 60℃, 30s; for 40 cycles.

[0065] ③Results Analysis:

[0066] qPCR standard curve of the target gene (e.g.) Figure 1 (As shown) was prepared from E. coli genomic DNA carrying the RP4 plasmid. The target gene was amplified by PCR using primers in Table 4. The PCR product was then transferred to competent cells. Positive clones were screened, and colonies were recultured to extract plasmid DNA. DNA content and purity were determined, and serial dilutions were performed. A standard curve (R4) was plotted based on copy number and Cr value. 2 >0.99). The copy number of a specific gene can be calculated using formula 1.1:

[0067] Copies / μL=(6.02×10 23 (×DNA concentration) / (650×10) 9 ×base length)(1.1)

[0068] 1.2.3 Detection of active species

[0069] (1) Qualitative analysis of reactive oxygen species was performed at room temperature using an electron paramagnetic resonance spectrometer (EPR, Bruker EMS-plus). DMPO was used as SO4. ·- ·OH and superoxide radicals (O2) ·- ) Scavenger, with TEMP as the primary agent1 O2 scavenger.

[0070] (2) Methanol (MeOH, 400 mM), tert-butanol (TBA, 400 mM), potassium dichromate (K2Cr2O7, 0.05 mM), L-histidine (L-His, 10 mM), superoxide dismutase (SOD, 20 U / mL), dimethyl sulfoxide (DMSO, 10 mM), and polyisopropyl acetone (PMSO, 20 mM) were used to identify sulfate radicals / hydroxyl radicals (SO4) in the system. ·- ), hydroxyl radical (·OH), electron (e) - ), singlet oxygen ( 1 O2), superoxide (O2) ·- SO4 bound to the surface ·- ·OH and high-valence metal oxides.

[0071] (3) Quantitative analysis of bioactive species

[0072] The catalyst was dispersed in a solution containing 0.25 mM terephthalic acid and 1 mM sodium hydroxide. 3 mL of the liquid was taken at each time point and filtered through a 0.22 μm filter membrane. Fluorescence intensity was measured using a fluorescence spectrometer (excitation wavelength 315 nm).

[0073] 1 mL of bacterial culture was mixed with 0.05 mM DPBF at each time point (0, 25, 50, 75, and 100 min) to quantitatively detect the PDS activation generated in the reaction system. 1 O2. The absorbance was measured at 411 nm using a UV-Vis spectrophotometer to calculate the concentration of DPBF.

[0074] 1.2.4 ARB Damage Analysis

[0075] (1) SEM observation: Bacterial cultures collected from the Mo / NC-400 activated PDS system at different time points were centrifuged at 10,000 rpm for 5 min, the supernatant was discarded, and the samples were washed three times with PBS buffer and the cells were resuspended. Subsequently, the samples were fixed on silicon wafers with 2.5% glutaraldehyde solution pre-cooled to 4℃ and stored overnight at 4℃. Then, the samples were dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol at 4℃ for 20 min each. Finally, the samples were lyophilized for 2 h, sputter-coated with gold, and observed by SEM.

[0076] (2) TEM observation: Samples were collected from the reaction system at different time points, centrifuged at 6000 rpm for 15 min, the supernatant was discarded, the precipitate was collected and washed twice with 0.85% NaCl. The collected E. coli solution was fixed with pre-cooled 2.5% glutaraldehyde solution for 2 h, stored with ice packs, and immediately sent to NS Company for ultrathin section preparation and TEM testing.

[0077] 1.2.5 Detection of cell membrane permeability and oxidative stress level

[0078] (1) Cell membrane permeability detection

[0079] Lactate dehydrogenase (LDH) levels were detected using a cytotoxicity assay kit from Beyotime Biotechnology Co., Ltd., with approximately 10 samples collected at each time point (0, 25, 50, 75, and 100 min). 8 CFU mL -1 1.5 mL of bacterial culture was processed. Lysis buffer was added, and the culture was sonicated (200 W, 3 s sonication, 10 s interval, repeated 50 times). The sonicated culture was centrifuged at 10000 × g for 10 min, and the supernatant was collected. 120 μL of the supernatant was mixed with 60 μL of LDH detection working solution and incubated at room temperature in the dark for 30 min. The absorbance was measured at 490 nm to quantitatively detect changes in LDH content.

[0080] (2) Intracellular reactive oxygen species detection

[0081] Using a bacterial reactive oxygen species detection kit O11 was used to detect reactive oxygen species levels, and samples containing approximately 10 mg / L were collected at 0, 25, 50, 75, and 100 minutes. 8 CFU mL -1 1.5 mL of fresh bacterial culture was centrifuged at 10000 × g for 5 min to collect the precipitate, and washed 2–3 times with PBS. The cells were then resuspended in 1 mL of staining solution and incubated at 37 °C in the dark for 30 min. After incubation, the cells were collected by centrifugation, washed once more with PBS, centrifuged again, and resuspended in 500 μL of PBS. The cells were thoroughly mixed, and 200 μL of the cell solution was added to a black 96-well plate for fluorescence detection using a microplate reader.

[0082] (3) Enzyme activity detection

[0083] Superoxide dismutase (SOD) and catalase (CAT) assay kits were used to determine their levels. Bacterial cultures at 0, 25, 50, 75, and 100 min were centrifuged at 10000×g for 5 min, and the precipitate was discarded. 500 μL of extraction buffer was added to the supernatant, and the mixture was sonicated (200 W, 3 s sonication, 10 s interval, repeated 50 times). The mixture was then centrifuged at 8000×g for 10 min at 4°C, and the supernatant was placed on ice for detection. Reagents were added sequentially according to the manufacturer's instructions, and the absorbance of each tube was measured using a microplate reader.

[0084] 1.2.6 ATP Activity Detection

[0085] ATP levels were determined using the Beyotime ATP assay kit, with 10 samples taken at 0, 25, 50, 75, and 100 minutes. 8 CFU mL -1 Add 1 mL of the treated bacterial culture, centrifuge at 8000 rpm for 5 min and discard the supernatant. Add 200 μL of lysis buffer, centrifuge again at 4 °C for 5 min (12000 × g), and discard the precipitate. Add 100 μL of supernatant and 100 μL of detection working solution to a black 96-well plate and mix well. All operations were performed on ice.

[0086] 1.3 ARGs conjugation and transfer experiments

[0087] 1.3.1 Binding Transfer Experiment

[0088] (1) Using Escherichia coli containing RP4 (supplemented with 50 mg / L Km, 40 mg / L Tc and 100 mg / L Amp) as donor bacteria, and Escherichia coli carrying rifampicin ARGs (E. coli, Rif) as recipient bacteria, the bacterial concentration was 10. 9 CFU mL -1 The donor-recipient bacterial ratio was adjusted to 1:1. The experiment was conducted in three scenarios: (i) 0.5 mL of treated donor bacteria was mixed with 0.5 mL of untreated recipient bacteria; (ii) 0.5 mL of untreated donor bacteria was mixed with 0.5 mL of treated recipient bacteria; and (iii) 0.5 mL of treated donor bacteria was mixed with 0.5 mL of treated recipient bacteria. The donor and recipient bacteria from each of these scenarios were cultured together for 24 h in liquid medium supplemented with 50 mg / L Km, 40 mg / L Tc, 100 mg / L Amp, and 100 mg / L Rif, respectively. Then, the medium was serially diluted 10-fold to appropriate concentrations, and conjugates were screened using the pour plate method on plates containing 100 mg / L Rif, 50 mg / L Km, 40 mg / L Tc, and 100 mg / L Amp. Plates containing 100 mg / L Rif were used as a control for selecting recipient bacteria. The plates were incubated at 37°C for 24 h, and the number of resistant colonies was counted.

[0089] 1.3.2 Quantitative Detection of Biofilm and Major Components

[0090] (1) Biofilm detection: In a 96-well plate, 20 μL of donor or recipient bacterial culture was inoculated onto 180 μL of LB liquid medium to form a single biofilm. 10 μL of donor bacteria and 10 μL of recipient bacteria were inoculated onto 180 μL of LB liquid medium in a 96-well plate to form a dual biofilm. 20 μL of sterile water was added to 180 μL of LB liquid medium as a control. The 96-well plate was incubated statically at 37 °C for 12 h. Biofilm biomass was quantified using the crystal violet optical method. After biofilm culture, the floating phase was removed, and each well was washed twice with PBS. Subsequently, 200 μL of methanol was added to each well to fix the attached biofilm for 15 min, and then the methanol was removed and the well was dried. 0.1% (200 μL, v / v) of crystal violet was added to each well, and the biofilm was stained and fixed in the dark at room temperature for 30 min, followed by washing three times with water to remove excess dye. Finally, the attached crystal violet was dissolved in 30% acetic acid (150 μL, v / v) solution at 37°C for 20 min. The absorbance of each well was measured at 590 nm using a microplate reader to determine the biomass of biofilm formation. The above experiment was repeated three times.

[0091] (2) Detection of extracellular polymeric substances (EPS): EPS was extracted from the three binding systems described above. First, 10 mL of bacterial culture was centrifuged at 5000 rpm for 10 min at 4 °C and washed twice with 0.9% (v / v) NaCl. Then, the bacteria were resuspended in 10 mL of PBS buffer and heated at 70 °C for 1 h. Subsequently, the culture was centrifuged at 12000 rpm for 15 min at 4 °C, and the supernatant was filtered through a 0.22 μm pore size filter to obtain EPS.

[0092] (3) Protein quantification: Take 20 μL of EPS for each time period as described in (2) above, add 200 μL of BCA detection working solution, incubate at 37°C for 30 min, and then use an ELISA reader to detect the absorbance at 562 nm.

[0093] (4) Determination of total polysaccharide content: Mix 1 mL of EPS sample with 1 mL of deionized water and 1 mL of 6% phenol solution in a 15 mL EP tube and vortex to mix. Then slowly add 5 mL of concentrated sulfuric acid to the side of the tube, vortex for 5 s, and incubate at room temperature for 30 min. Cool all tubes to room temperature and then read the absorbance at 490 nm using a UV-Vis spectrophotometer.

[0094] (5) Extraction of eDNA using ethanol precipitation: Mix 10 mL EPS, 1 mL sodium acetate, and 22 mL pre-cooled ethanol thoroughly. Place the sample at -20℃ for 30 min to precipitate the DNA, then centrifuge at 10,000 rpm for 20 min at 4℃, and carefully collect the supernatant. After the ethanol has completely evaporated, add an appropriate amount of ddH2O to dissolve the DNA precipitate, and determine the eDNA concentration using a micro-volume UV-Vis spectrophotometer.

[0095] 1.3.3 Transcriptome Sequencing

[0096] 200 mL of donor bacterial treatment solution was collected at 0, 30, and 60 min intervals and centrifuged at 10000×g for 2 min at 4 °C to obtain cell pellets. The pellets were then rapidly frozen in liquid nitrogen for 15 min and stored at -80 °C. The mRNA expression levels were then analyzed at CAS New Life Biotechnology Co., Ltd.

[0097] 2. Results and Discussion

[0098] 2.1.1 Effect of calcination temperature on sample morphology and structure

[0099] To investigate the effect of different calcination temperatures on the catalyst, this study obtained three catalysts, Mo / NC-400, Mo / NC-500, and Mo / NC-600, by calcination at 400, 500, and 600 °C, respectively. Figure 2 As shown in Figure ab, HRTEM observation revealed that Mo / NC-400 exhibits a plate-like structure with no nanoparticle aggregation. The morphology of Mo / NC at different temperatures was compared. Figure 2 In the C-C layer, it can be observed that although both exhibit a lamellar structure, the lamellar thickness increases with increasing temperature. Further analysis using HAADF-STEM reveals that Mo is dispersed as single atoms on the C layer. Figure 2 The bright spots circled in yellow in the middle represent individual Mo atoms. Figure 2 EDS elemental analysis of the sample confirmed that C, N and Mo are uniformly distributed in Mo / NC-400.

[0100] like Figure 3 As shown in Figure a, the XRD results of Mo / NC-400, Mo / NC-500, and Mo / NC-600 show two characteristic peaks at 25.3° and 44.5° respectively. However, the intensity of both peaks decreases with increasing calcination temperature, possibly due to changes in the lamellar structure of the catalyst. Figure 3 b in the image shows the Raman spectrum of the sample. All catalysts were analyzed at 1350 cm⁻¹. -1 and 1590cm -1 There are two main overlapping spectral bands nearby, among which the Mo / NC-400 structure exhibits the highest degree of disorder (ID / I G =1.05), and its abundant structural defects provide more active sites for the reaction.

[0101] XPS was used to analyze the surface chemical composition of catalysts calcined at different temperatures. For example... Figure 3 As shown in Figure c, all three catalysts contain the elements C, Mo, and N. Figure 3 In the figure, d represents the high-resolution C1s spectrum of the sample. As can be seen, three peaks were observed at 284.8, 286.1, and 289.1 eV for Mo / NC-400, corresponding to the CC / C=C, CN, and C=O groups, respectively. With increasing temperature, the binding energy of C1s shifted slightly towards higher binding energies, which may be related to changes in the C structure. The CN content decreased with increasing calcination temperature, indicating that high temperatures may damage the N-doped structure. The N 1s spectrum is shown below. Figure 3 As shown in Figure e, Mo / NC-400 exhibits binding energies at 396.8, 398.1, 399.6, and 402.1 eV; Mo / NC-500 shows four characteristic peaks near 396.8, 398.4, 400.6, and 402.5 eV; and Mo / NC-600 shows four characteristic peaks near 396.8, 398.6, 399.9, and 402.5 eV, which are attributed to Mo-N, pyridine N, pyrrole N, and N oxide, respectively. Pyridine N and pyrrole N contribute to the stability of Mo atoms on the carbon framework. The content of pyrrole N and N oxide increases with increasing calcination temperature, possibly because pyridine N transforms into the more stable pyrrole N and N oxide. Figure 3 As shown in Figure f, Mo 3d of Mo / NC-400 exhibits four characteristic peaks at 231.4, 232.90, 235.6, and 236.0 eV, with 232.90 and 236.00 eV attributable to Mo. 6+ 231.4 and 235.6 eV belong to Mo 4+ This indicates that the sample contains Mo. 6+ and Mo 4+ Coexistence. The Mo 3d binding energies of both Mo / NC-500 and Mo / NC-600 changed compared to Mo / NC-400, indicating that different calcination temperatures altered the microenvironment of the metal sites in the catalysts.

[0102] 2.2 The inactivation effect of bacteria and its causes

[0103] 2.2.1 Bacterial inactivation effect

[0104] Using *E. coli* carrying the RP4 plasmid as a model bacterium, the inactivation effects of catalysts at different calcination temperatures on the bacteria were compared. Figure 4As shown in Figures ab, the catalyst alone has almost no effect on inactivating *E. coli*. When PDS and the catalyst are present together, Mo / NC-400, Mo / NC-500, and Mo / NC-600 all exhibit sterilization performance, reducing bacterial counts by 7.3 log, 6.4 log, and 5.6 log bacteria, respectively, within 100 min. These results indicate that Mo / NC-400 has the best sterilization effect, possibly due to the change in the N coordination number, which alters the catalyst's coordination environment and enhances its catalytic activity.

[0105] Although Mo / NC-400 is effective in killing ARBs, its extent of degradation of bacterial DNA and ARGs is unclear. Therefore, the degradation effect of the optimal material Mo / NC-400 on these bacteria was further investigated. The results are as follows: Figure 4 As shown in Figure c, the total DNA concentration decreased with increasing reaction time, and the total DNA removal rate was 87.64% within 100 min. Figure 4 Figure d shows the degradation effect of Mo / NC-400 on the target genes. After 100 min of reaction, the amounts of the target genes tetA, APH-Ib, blaTEM-2, and the housekeeping gene 16S rRNA decreased by 5.23 log, 4.94 log, 4.26 log, and 3.86 log, respectively. Figure 4 As shown in b, Mo / NC-400 can not only efficiently inactivate ARBs, but also effectively degrade ARGs, thereby inhibiting the spread of ARGs.

[0106] 2.2.2 Effects of Mo / NC-400 activated PDS on bacterial oxidative damage

[0107] To further investigate the oxidative damage process of bacterial cells during Mo / NC-400 sterilization, the activities of several related enzymes were monitored. For example... Figure 5 As shown in Figure a, with prolonged treatment time, the accumulation of intracellular ROS in Mo / NC-400 cells continuously increased, reaching a maximum at 75 min. When this exceeded the stress tolerance of the bacterial defense system, the cells gradually ruptured and eventually died, resulting in the outflow of intracellular ROS and a significant decrease in ROS levels. SOD and CAT are two antioxidant enzymes that act as scavengers of bioactive species, effectively protecting cells from oxidative damage induced by these species. Therefore, the intracellular SOD and CAT levels were measured. Figure 5As shown in Figure b, the activities of SOD and CAT initially increased and then decreased during sterilization. This is because a large number of bioactive species are produced and continuously attack the cells in the early stages of sterilization, causing oxidative stress in bacteria and leading to an increase in the activity of antioxidant enzymes. As the bioactive species accumulate, their production exceeds the load of the bacterial stress system, causing severe damage or even death to *E. coli* cells, resulting in a decrease in antioxidant enzyme activity. After the oxidative defense system collapses, the bacteria begin to attack the bacterial cell membrane. The outer membrane of *E. coli* cells mainly functions to maintain bacterial morphology and balance osmotic pressure; removing lipids from the cell membrane impairs its functional integrity, thus inhibiting bacterial activity or causing death. Therefore, we further investigated the release of lactate dehydrogenase (LDH) during bacterial inactivation. Since the greater the cell membrane permeability, the greater the LDH release, changes in cell membrane permeability can be determined by analyzing the amount of LDH released. Figure 5 As shown in Figure c, the amount of LDH released gradually increases with the extension of reaction time, indicating that the active species attack the bacteria, leading to a gradual increase in cell membrane permeability and a gradual loss of bacterial activity.

[0108] 2.2.3 Effects of Mo / NC-400 activated PDS on bacterial cell membrane permeability

[0109] Changes in the cell membranes of *E. coli* bacteria before and after sterilization were observed using SEM and TEM. Figure 6 As shown in Figure 1, untreated E. coli cells are plump with intact and smooth outer membranes; however, after 100 minutes of treatment, the bacteria shrank, and the cell membranes became rougher and even ruptured, leading to the outflow of cytoplasm and contents. This further demonstrates that the Mo / NC-400 activated PDS system has a disruptive effect on bacterial cell membranes.

[0110] 2.3 Study on the mechanism of catalyst activation of PDS

[0111] 2.3.1 Identification of active species in the Mo / NC-400 activated PDS system

[0112] To determine the possible sterilization mechanism of *E. coli*, the consumption of PDS was analyzed and a premixing experiment was conducted. 100 μL of samples from different time points were added to 10 mL EP tubes, followed immediately by 4.9 mL of a solution containing 0.4150 g KI and 0.0200 g NaHCO3. The mixture was mixed, allowed to stand for approximately 15 min, and then measured at 352 nm using a UV / Vis spectrophotometer. The results are shown below. Figure 7 As shown in Figure a, it can be observed that the amount of PDS in the system decreases continuously with the extension of reaction time, indicating that PDS is continuously consumed during the reaction. Since the bioactive species in the system have a short lifespan, we speculate that adding E. coli after Mo / NC-400 reacts with PDS for a period of time will likely reduce the sterilization effect. Figure 7As shown in Figure b, the premixing experiment results indicate that the sterilization effect was not affected when the premixing time was 20 min and 40 min; however, when the premixing time was extended to 60 min and 80 min, the number of E. coli decreased to 2.8 and 3.7 log CFU / mL, respectively. -1 In summary, the active species generated during PDS activation play a major role in the Mo / NC-400 activated PDS system during sterilization.

[0113] Based on the above results, EPR was used to detect and analyze the active species in the Mo / NC-400 activated PDS system. DMPO was added to the sterilization system as ·OH and SO4. ·- Capture agents, such as Figure 7 As shown in Figure c, a signal peak of 1:2:1:2:1:2:1 appears, which is a characteristic peak of DMPO oxidation, and the signal intensity becomes stronger as the reaction time increases. Figure 7 In the same way, DMPO is used as O2. ·- The trapping agent was used, but no signal was detected. TEMP was used as... 1 O2 scavengers, such as Figure 7 As shown in Figure e, a signal with a characteristic peak of 1:1:1 was observed, and the signal intensity increased with the extension of reaction time, indicating the presence of non-free radicals in the sterilization system. 1 The presence of O2 confirms that the active substances in the Mo / NC-400 activation reaction of PDS include ·OH and SO42-. ·- and 1 O2. This could be because of O2. ·- Unstable, usually transforms into 1 O2 exists in the system. Therefore, SOD can be used as a source of O2. ·- The cleaning agent was then tested with EPR. 1 Changes in O2, such as Figure 7 As shown in f, after adding SOD 1 The weakening of the ternary signal peak of O2 is consistent with our prediction, indicating that O2 in the system... ·- Quickly converted into 1 O2.

[0114] To further analyze which active species in the system play a role in the sterilization process, this study used MeOH, TBA, K2Cr2O7, L-His, SOD, DMSO, and PMSO as ·OH and SO42-, respectively. ·- e - , 1 O2, O2 ·- Surface-bound free radicals and high-valence metal oxide scavengers. For example... Figure 8As shown in Figure a, the addition of L-His did not significantly change the number of E. coli, indicating that there was virtually no ARB inactivation. 1 O2 plays a major role in the sterilization process. Because 1 The half-life of O2 in D2O is more than 10 times longer than that in water. Therefore, using D2O instead of water as a solvent resulted in a better sterilization effect, killing all ARBs within 75 minutes, further demonstrating... 1 O2 plays a role in sterilization. The addition of MeOH significantly inhibited the sterilization effect, indicating that SO42-... ·- SO4 plays an important role in the inactivation of ARB. ·- Not only can it directly cause ARB death, but it can also indirectly generate ·OH oxidation attack on ARBs. After adding SOD or TBA, the number of ARBs also decreased, but the inhibitory effect was significantly lower than that of L-His and MeOH, indicating that O2... ·- ·OH plays a role in the sterilization process. Adding K₂Cr₂O₇ and DMSO to the sterilization system had almost no effect on the inactivation effect, indicating that the presence of ·OH in the system... - SO4 bonded to the surface ·- The sterilization effect of ·OH radicals is negligible. Besides free radicals, high-valence metal oxides also possess sterilization capabilities, so PMSO was used to capture these oxides in the system. It was found that PMSO had no effect on the sterilization ability of the Mo / NC-400 activated PDS system. Since PMSO can be oxidized by high-valence metal oxides to form PMSO2, high-performance liquid chromatography (HPLC) was used to detect changes in the PMSO and PMSO2 content in the system. Figure 8 As shown in Figure b, the PMSO content did not decrease within 100 minutes, and PMSO2 was not detected, indicating that high-valence metal oxides were not present in the system. Since dissolved oxygen in water can also be converted into free radicals, to determine the effect of dissolved oxygen in the aqueous solution on the sterilization effect, N2 was introduced into the Mo / NC-400 / PDS system to remove O2 from the water. Figure 8 As shown in Figure c, the inactivation of Escherichia coli after the addition of N2 did not change compared with the control group, indicating that dissolved oxygen did not affect the sterilization effect of Mo / NC-400 / PDS.

[0115] Further, the system was modified using 1,3-diphenylisobenzofuran and terephthalic acid. 1 Quantitative analysis of O2 and ·OH was performed. Because DPBF can react with… 1 O2 reacts in a 1:1 molar ratio to form an internal peroxide, which further decomposes into 1,2-dibenzoylbenzene, causing a decrease in the absorption peak of DPBF at 411 nm. Therefore, UV-Vis detection of DPBF can be used to determine the presence of DPBF in the system. 1 The content of O2. For example... Figure 9 As shown in Figure a, 1 The O2 content increases with increasing reaction time, a result consistent with EPR analysis. Furthermore, ·OH can react with terephthalic acid to form 2-hydroxyterephthalic acid; the formation of 2-hydroxyterephthalic acid is detected using a fluorescence spectrophotometer, indirectly determining the concentration of ·OH in the system. Figure 9 As shown in Figure b, the ·OH content increases with increasing reaction time. This indicates the presence of ·OH in the system. 1 O2 and ·OH.

[0116] 2.3.2 Electrochemical Analysis of the Mo / NC-400 Activated PDS System

[0117] Studies have shown that contact between the catalyst surface and PDS promotes pollutant degradation through electron transfer. To determine whether electron transfer exists in the Mo / NC-400 activated PDS system, linear voltammetry (LSV) and chronoamperometry (it) were used to investigate electron transfer in the system. Figure 10 As shown in Figure a, the LSV experimental results show that when only Mo / NC-400 is present, there is only a weak current in the system; after adding PDS, the current response increases significantly, which is the result of electron transfer between PDS and Mo / NC-400; when PDS coexists with E. coli, the current response is further enhanced, indicating that there is electron transfer between E. coli and PDS in the system. Figure 10 More definitive evidence can be found through the it curve in section b. The sequential addition of PDS and E. coli to the electrolyte solution both resulted in an increase in the current signal. However, compared to PDS, the change in current signal after the addition of E. coli was lower, indicating that electron transfer mainly occurred between the PDS and the Mo / NC-400 surface.

[0118] 2.3.3 Determination of catalyst active sites

[0119] Studying active sites is beneficial for understanding the activation mechanisms of reactants and the catalytic behavior of active sites at the atomic level, and helps to reveal the reaction mechanism between single-atom catalysts and reactants. To identify active sites in the catalyst, KSCN was first used as a scavenger for Mo-N sites, such as... Figure 11As shown in Figure a, the sterilization effect of all three catalysts was significantly inhibited after the addition of KSCN. The Mo / NC-400, Mo / NC-500, and Mo / NC-600 systems showed inactivations of 1.9 log, 5.2 log, and 2.0 log E. coli, respectively, with the greatest inhibitory effect observed in Mo / NC-400. However, some E. coli remained, suggesting that other active sites may exist in the catalyst besides Mo-N sites. Since H3PO4 can mask pyridine N sites, and the catalyst treated with H3PO4 can recover pyridine N sites after soaking in K2CO3, this method was chosen to further determine whether pyridine N active sites exist in the catalyst. Figure 11 As shown in Figure bd, the sterilization effect of the catalyst was inhibited after treatment with H3PO4, but the sterilization effect was completely restored after treatment with K2CO3. These results indicate that the catalyst contains two active sites: Mo-N and pyridine N.

[0120] In summary, the active sites of Mo / NC-400 are Mo-N and pyridine N. These active sites adsorb PDS, thereby activating PDS to produce... 1 O2, SO4 ·- Electrochemical analysis revealed electron transfer among the catalyst, E. coli, and PDS, primarily occurring between the catalyst and PDS. This indicates that Mo / NC-400 adsorbs PDS through its active sites, leading to electron transfer between the two and resulting in the activation of PDS and the generation of the main active species. 1 O2.

[0121] 2.4 Analysis of the reasons why catalysts inhibit ARGs binding and transfer

[0122] 2.4.1 Changes in ARGs binding transfer frequency in the Mo / NC-400 activated PDS system

[0123] To investigate the effects of donor and recipient bacteria on conjugation transfer, the changes in ARGs conjugation transfer frequency were studied under three conditions: treatment with only donor bacteria, treatment with only recipient bacteria, and simultaneous treatment with both donor and recipient bacteria. Figure 12 As shown in Figure a, when only the donor bacteria are treated, the number of recipient bacteria remains unchanged. When only the recipient bacteria are treated, the number of recipient bacteria decreases from 9.0 log to 5.2 log. When both are treated simultaneously, the number of recipient bacteria decreases to 5.9 log, which is higher than the number of bacteria treated with recipient bacteria alone. This is because the donor and recipient bacteria have different tolerance levels, and the catalyst is more likely to attack the intolerant donor bacteria. Figure 12 Figure b shows the number of conjugates. As can be seen from the figure, when only the recipient bacteria are treated, the number of conjugates is the lowest from 15 min to 60 min, indicating that the number of recipient bacteria has a greater impact on conjugation transfer. Figure 12In the figure, c represents the conjugation transfer frequency. When only the donor bacteria are treated, the conjugation frequency gradually decreases with increasing reaction time, and no conjugation transfer occurs at 60 min. When only the recipient bacteria are treated and both donor and recipient bacteria are treated simultaneously, the conjugation transfer frequency shows a trend of first increasing and then decreasing, with a maximum value of 3.5 × 10⁻⁶. -6 and 2.7×10 -6 Although the bonding frequency increases, the number of bonding molecule decreases. Therefore, it is still believed that Mo / NC-400 can suppress bonding transfer of ARGs.

[0124] 2.4.2 Detection of relevant indicators of donor and recipient bacteria

[0125] Accumulation of intracellular ROS in ARB cells increases cell membrane permeability, triggering the SOS response and thus promoting ARG conjugation and transfer. This study examined changes in intracellular ROS levels in donor and recipient bacteria, such as... Figure 13 As shown in Figure a, intracellular ROS gradually accumulates in the first 15 minutes, but the ROS level decreases after 15 minutes. This is because the accumulated ROS exceeds the ARB's own defense capacity, leading to cell damage and lysis, resulting in ROS efflux and a decrease in ROS content. While excessive ROS accumulates in E. coli cells, it also attacks the ARB cell membrane, causing lipid peroxidation, resulting in membrane structure damage and impaired cell function. Malondialdehyde (MDA) is the end product of lipid peroxidation, and its changes can reflect the degree of lipid loss in E. coli cells. Figure 13 As shown in Figure b, the intracellular MDA content of both donor and recipient bacteria increased with increasing reaction time. Higher MDA content indicated more severe lipid peroxidation, suggesting that Mo / NC-400 causes cell membrane damage. This reflects that continuous oxidative stress during sterilization leads to increased lipid peroxidation of the cell membrane, thereby damaging cell membrane integrity and increasing cell membrane permeability. ATP is one of the limiting factors for conjugation transfer, providing energy for DNA transfer and inducing conjugation transfer. This study detected changes in intracellular ATP content, and the results are shown below. Figure 13 As shown in Figure c, the ATP content first increases and then decreases. The increase in ATP content in the first 15 minutes may be to provide energy for DNA transfer during conjugation. The subsequent continuous decrease in ATP content may be related to the decrease in the number of recipient bacteria, which leads to a decrease in the frequency of conjugation.

[0126] Escherichia coli (E. coli) is a common opportunistic pathogen found in the intestines of humans and animals. Biofilms are the primary hosts for ARGs (autotrophic globulins) and provide favorable conditions for horizontal transfer. The main components of biofilms include extracellular polymers (EPS), primarily proteins, polysaccharides, and eDNA. Therefore, this study examined the changes in biofilms and EPS (proteins, polysaccharides, and eDNA) of donor and recipient bacteria under Mo / NC-400 treatment. The results are as follows: Figure 14As can be seen from Figure a, the biomass of the biofilm decreases as the treatment time increases. Figure 14 The levels of protein, polysaccharide, and eDNA in the bd cells were all lower than before Mo / NC-400 treatment. Based on the above, it is speculated that Mo / NC-400 treatment may activate bacterial oxidative stress, enhance cell membrane permeability, and inhibit ATP synthesis and biofilm formation.

[0127] 2.4.3 Expression of donor bacteria-related genes

[0128] Studies have shown that ARG conjugation transfer is more dependent on donor bacteria. Therefore, this study performed transcriptome sequencing analysis on donor bacteria treated with Mo / NC-400 at three time points: 0, 30, and 60 min. The results are as follows: Figure 15As shown, the downregulation of expression of genes related to conjugation transfer (ftsY, tesB) indicates that conjugation transfer was inhibited. Since SOD and CAT are important antioxidant enzymes for removing ROS, the downregulation of sodA and sodB in the treatment groups (30 min and 60 min) compared to the control group may be due to DNA damage caused by excessive ROS accumulation. The upregulation of genes related to ROS generation (katE, azoR, zntA, rsxC, and rsxA) by 1.3–1.9-fold indicates that the ARB antioxidant system was disrupted, inducing oxidative stress in ARBs. The SOS response mainly protects cellular DNA and promotes conjugation transfer by disrupting genes encoding conjugation transfer inhibitors and upregulating the expression of plasmid transfer-related genes. Genes related to the SOS response (dinI, rpnC, rpnA, soxS, cho, recR, recQ, and recF) were upregulated by 1.1–1.9-fold, and genes related to DNA repair (rpoH, dnaA, and dnaN) were also upregulated, further demonstrating that ROS production and the SOS response are activated in the conjugation transfer system. Excessive ROS leads to cell fragmentation and lysis, thereby affecting DNA repair capabilities. Conjugation transfer is closely related to the integrity of the cell membrane between ARBs; cell membrane damage inhibits the conjugation transfer of ARGs. Analysis of cell membrane-related gene expression revealed decreased expression levels of lpp, fhuA, ompA, ompX, ompW, bamB, osmB, osmE, blc, slp, exbB, and exbD genes. The lpp gene maintains cell structure and is associated with bacterial biofilm formation; the fhuA gene is associated with bacterial outer membrane structural integrity and antibiotic sensitivity; the omp family in *E. coli* protects ARBs from damage, maintaining cell membrane stability and integrity; the bamB gene encodes outer membrane protein transport; osmE and osmB play important roles in maintaining cell membrane osmotic pressure; the blc gene encodes lipid transport proteins on the cell membrane, regulating bacterial immunosuppression; slp is an outer membrane protein that can stabilize outer membrane structure through carbon starvation, playing a crucial role in protecting ARBs from external stimuli; the exbB and exbD genes are involved in bacterial energy transfer and transmembrane transport, and decreased expression of these genes indicates that cell membrane integrity is affected. These gene expression changes suggest that Mo / NC-400 treatment inhibits the normal functional structure of the ARB cell membrane, increases cell membrane permeability, enhances cell membrane lipid peroxidation, and ultimately leads to ARB death.

[0129] The energy metabolism pathways of *E. coli* include the glucose-phosphotransferase system, glycolysis and gluconeogenesis, pyruvate metabolism, the tricarboxylic acid cycle (TCA), and the electron transport chain (ETC). Transcriptome analysis was used to analyze the expression of genes related to these energy metabolism pathways in *E. coli*, and the results are as follows: Figure 16As shown, the decreased expression of genes related to ATP synthesis (atpA, atpC, atpE, and atpG) confirms the reduction in ATP synthesis within the bacteria. The expression of genes related to glucose precursor synthesis (deoB, pgL, and tktA) and genes related to glycolysis (pfkA, pykB, crr, pgi, ahr, yiaY, yahK, gpmA, pykF, and pykA) was simultaneously downregulated. Since glucose is the main carbon source in *E. coli* metabolism, the weakened expression of related genes suggests that reduced intracellular glucose levels may affect carbon source metabolism efficiency. Downregulation of the phosphotransferase system genes crr and ptsG may inhibit glucose transmembrane transport, thus exacerbating carbon source depletion. The phosphofructokinase encoded by pfk is the rate-limiting enzyme in glycolysis and plays a crucial role in the conversion of glucose into a carbon source. The pyruvate kinase encoded by pyk is located at a major metabolic crossover point, catalyzing the production of pyruvate and ATP. Pyk is a key enzyme in the glycolysis pathway, catalyzing the conversion of phosphoenolpyruvate to pyruvate. Downregulation of the expression of both pfk and pyk genes leads to a decrease in the rate of glucose breakdown, inhibiting the conversion of phosphoenolpyruvate to pyruvate, and ultimately significantly reducing the supply of ATP synthesis. Downregulation of pyruvate pathway-related genes (ackA, adhE, yahK, adhP, yccX, pflB, poxB, and ldhA), particularly the reduced activity of acetyl-CoA convertase encoded by the ackA gene, not only reduces acetic acid production but also inhibits NAD+ regeneration (this reaction generates 2 NAD+ molecules for every 1 molecule of pyruvate converted). Low expression of lactate dehydrogenase (ldhA) and aldehyde dehydrogenase (adhE) indicates that the compensatory pathway of lactate / ethanol fermentation has not been activated. This dual inhibition indicates that under restricted glycolysis, cells cannot maintain the NAD+ / NADH balance through anaerobic fermentation, exacerbating the energy crisis. The expression of TCA cycle-related genes (aceE, aceF, and fumD) is downregulated, while the expression of lpd, the gene encoding lipamide dehydrogenase, is upregulated. lpd catalyzes the oxidation of α-ketoglutarate to succinyl-CoA and NADH; its upregulation may promote succinyl-CoA accumulation, driving the diversion of energy-rich intermediates towards fatty acid synthesis. Reduced TCA cycle efficiency inhibits the oxidative consumption of acetyl-CoA, decreasing the supply of NADH / FADH2 into the electron transport chain, while simultaneously limiting NAD+ regeneration, further disrupting redox homeostasis. The electron transport chain (ETC) includes complexes I–IV, the electron transport protein ubiquinone, and cytochrome C. Electron flow couples with the generation of a proton gradient across the inner membrane, and the resulting energy is used by complex V (ATP synthase) to produce ATP. Complex I involves four genes (nuoM, nuoN, nuoL, and nuoJ), and complex II involves four genes (sdhA, frdA, frdC, and frdD).Upregulation of genes related to Complex I may be due to the inhibition of glycolysis leading to reduced NADH production, which in turn optimizes limited substrate utilization by improving electron transport efficiency. Downregulation of Complex II indicates suppression of the succinate-ubiquinone redox reaction, resulting in reduced electron input. Low expression of genes related to Complex IV (cydB and cydD) directly hinders terminal electron transport and proton gradient formation, weakening the driving force of oxidative phosphorylation. In summary, Mo / NC-400 inhibits glycolysis-pyruvate metabolism, blocks the synergistic energy supply of the TCA cycle and ETC through multiple mechanisms, and dismantles the emergency compensation mechanism of anaerobic fermentation, ultimately leading to a significant decrease in ATP synthase activity and a sustained reduction in intracellular ATP levels. This global collapse of the energy metabolism network may be a key molecular reason for its inhibition of conjugation transfer.

[0130] Quorum sensing (QS) plays a crucial role in bacterial biofilm formation, further influencing the spread of ARB resistance. Therefore, the expression of genes related to quorum sensing (QS) and biofilm formation was analyzed, such as... Figure 17 As shown, downregulation of genes related to QS (pdeR, kdpE, gadA, gadB, and gadC), biomembrane (csrA), polysaccharides (lptA, lptB, lptE, fadL, and fadM), and proteins (xylH, ugpB, ftsX, artP, artI, ccmA, and fepC) was observed. Based on this, it is inferred that Mo / NC-400 can inhibit ARB biomembrane formation, thereby inhibiting ARG conjugation and translocation. Based on the above analysis, it is hypothesized that Mo / NC-400 reduces conjugation and translocation by inducing ARB oxidative stress, activating SOS defense, enhancing ARB cell membrane permeability, disrupting ATP synthesis pathways, and inhibiting biomembrane formation.

[0131] By constructing a conjugation transfer model, this invention demonstrates that Mo / NC-400 can effectively inhibit the conjugation transfer of ARGs. Combined with gene transcriptome sequencing technology, it was revealed that Mo / NC-400 inhibits the conjugation transfer of ARGs by inducing oxidative stress in donor bacteria, activating the SOS response, enhancing ARB cell membrane permeability, inhibiting ATP synthesis and reducing energy supply, and disrupting ARB biomembrane formation.

[0132] 3. Conclusion

[0133] This invention prepares a single-atom catalyst that can efficiently activate PDS by altering the Mo coordination environment through calcination. The catalytic activity of Mo / NC was evaluated using ARBs and ARGs as target pollutants. The inactivation mechanism of ARBs was analyzed in detail through oxidative stress and cell membrane permeability, and the activation mechanism of PDS was proposed through active site and active species analysis. Finally, an ARGs conjugation and transfer model was constructed to analyze the horizontal transfer frequency of ARGs under conditions of donor bacteria treatment only, recipient bacteria treatment only, and simultaneous treatment of donor and recipient bacteria. Transcriptome sequencing revealed the reason why this system inhibits the horizontal transfer of ARGs. This study provides basic data and technical support for the application of reducing ARBs and ARGs in water bodies. Specific research conclusions are as follows:

[0134] (1) Mo / NC was prepared by high-temperature calcination. Mo was dispersed on the C-based material in the form of single atoms. The change of calcination temperature caused changes in the microenvironment around the Mo-N sites. The disorder of Mo / NC provided more active sites for the reaction.

[0135] (2) Mo / NC-400 activated PDS system can inactivate 7.3 log CFU / mL within 100 min. -1 The ARB. During sterilization, the catalyst can effectively adsorb PDS, and the transfer of electrons to PDS leads to its activation, thereby generating a large amount of... 1 O2 and SO4 ·- These bioactive species continuously attack bacteria, causing oxidative stress and increasing intracellular SOD and CAT concentrations to counteract the attack. When intracellular ROS levels exceed the tolerance of the bacteria's defense system, the antioxidant system is damaged, SOD and CAT concentrations decrease, and the cells are unable to effectively clear intracellular ROS, leading to cell death.

[0136] (3) In this system, the target genes tetA, APH-Ib, blaTEM-2, and the housekeeping gene 16S rRNA were degraded by 5.23 log, 4.94 log, 4.26 log, and 3.86 log, respectively. An ARGs transfer model was constructed to analyze the horizontal transfer frequency of ARGs under three conditions. When only the donor bacteria were treated, conjugation transfer was completely inhibited; when only the recipient bacteria were treated, or when both donor and recipient bacteria were treated simultaneously, the conjugation transfer frequency showed a trend of first increasing and then decreasing. Based on analyses of oxidative stress, cell membrane permeability, ATP synthesis, and transcriptome sequencing, the reasons for the inhibition of ARGs conjugation transfer were revealed: oxidative stress is generated during ARB inactivation, cell membrane permeability is damaged, energy metabolism is inhibited, and ATP synthesis is limited, ultimately leading to a decrease in the conjugation transfer frequency.

Claims

1. A method for inhibiting conjugation transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system, characterized in that: The Mo single-atom catalyst activates the persulfate system by activating oxidative stress response, disrupting cell membrane permeability, interfering with energy metabolism, and inhibiting biomembrane formation and resistance gene binding and transfer. The Mo single-atom catalyst is a Mo / NC catalyst, which is prepared by calcining anhydrous calcium citrate at 700-900℃ to form C material, then mixing the C material with ammonium molybdate and ammonium chloride to form a precursor, and calcining it at 400-600℃.

2. The method for inhibiting conjugation transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system according to claim 1, characterized in that: The Mo monatomic catalyst can efficiently activate peroxysulfate to generate active species 1 O2 and SO4 •− , and effectively remove antibiotic-resistant bacteria and antibiotic-resistant genes in water bodies, and in the reaction process, the active species induce antibiotic-resistant bacteria oxidative stress to trigger SOS response, enhance antibiotic-resistant bacteria cell membrane permeability, inhibit ATP synthesis path and reduce energy supply, thereby inhibiting the conjugative transfer of antibiotic-resistant genes.

3. The method for inhibiting conjugation transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system according to claim 2, characterized in that: The antibiotic resistance gene is tetA Gene, APH-Ib Genes and blaTEM-2 Gene.

4. The method for inhibiting conjugation transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system according to any one of claims 1-3, characterized in that: Application of the Mo single-atom catalyst-activated persulfate system in the preparation of drug-resistant pollution control agents for aquatic environments.

5. The method for inhibiting conjugation transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system according to claim 1, characterized in that... The specific preparation process of the Mo single-atom catalyst is as follows: Anhydrous calcium citrate is calcined in a tube furnace at a heating rate of 2-5℃ / min to 700-900℃ for 1-2 hours under nitrogen atmosphere. The calcined powder is thoroughly ground and stirred overnight in hydrochloric acid. It is then washed with ultrapure water until the solution is neutral and dried to obtain C material. 0.1924g of the C material prepared above is dispersed in 10mL of an aqueous solution containing 0.0144g of ammonium molybdate and 0.5000g of ammonium chloride. The solution is stirred at 80-90℃ until completely dry, then transferred to a tube furnace and calcined at 400-600℃ at a heating rate of 2-5℃ / min under nitrogen atmosphere for 2-3 hours. The solution is then naturally cooled to room temperature to obtain a Mo single-atom catalyst with a Mo mass percentage of 4%.

6. The method for inhibiting conjugation transfer of resistance genes based on a Mo single-atom catalyst-activated persulfate system according to claim 5, characterized in that: The preferred calcination temperature is 400℃.

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