电化学氧化同时去除抗性微生物和抗性基因及机制研究

【作者】 李伟；

【导师】 张弓；

【作者基本信息】 青岛大学 ， 微生物学， 2025， 硕士

【摘要】 抗生素耐药性（AMR）的全球蔓延已成为21世纪公共卫生领域的重大威胁。世界卫生组织（WHO）数据显示,2019年全球约127万人直接死于耐药菌感染,其中耐甲氧西林金黄色葡萄球菌（MRSA）因其多重耐药性（对β-内酰胺类、大环内酯类等7类抗生素耐药）和高致死率（血流感染死亡率达20-40%）,位列"超级细菌"清单首位。传统水处理技术（如氯化、紫外线）虽能部分灭活病原菌,但对携带抗性基因（ARGs）的耐药菌（ARB）去除效率有限,甚至可能通过诱导DNA损伤修复机制加速耐药基因的水平转移（HGT）。针对这一挑战,本研究创新性地构建了基于改性钯钛电极的循环式电化学氧化（ElectrochemicalOxidation,EO）系统,系统探究了其对临床重要耐药菌MRSA（ATCC43300菌株）及其耐药基因簇（SCCmec和mecA）的同步去除效能与分子机制。通过焦耳热技术对钛基阴极进行钯颗粒修饰,结合IrO₂/RuO₂涂层钛阳极,构建流通式反应装置（电极面积9cm²,极间距6 mm）。SEM-EDS表征显示,改性阴极形成多孔网状结构,比表面积较未修饰电极大,XPS证实表面存在PdO/PdO₂异质结（结合能335.8 eV和341.3 eV）,显著增强氯析出反应活性（Tafel斜率降低至67.4 mV/dec）。在模拟医疗废水（30 mM NaCl,pH 7.4）中,系统考察电流密度（1-6 mA/cm²）、电解液浓度（10-100 mM）等参数对MRSA灭活动力学的影响。结果表明:当电流密度达5m A/cm²时,活性氯（HClO/ClO^-）生成迅速,5分钟内实现7-log菌体灭活(从10⁷CFU/mL降至检测限以下。电极间距从12 mm缩小至6 mm可使单位体积反应活性位点密度提升40%。通过多尺度表征技术揭示EO对MRSA的致死路径:（1）透射电镜显示,处理3分钟后细胞壁出现10-50 nm裂隙,膜脂双层结构崩解（图3a）;（2）SYTO9/PI双染证实,5分钟处理使膜完整性丧失的细胞比例从0.5%升至99.7%;（3）离子色谱检测到胞内钾离子泄漏量从13 mg/L激增至32 mg/L（增幅146%）,LDH酶活性下降85%（p<0.01）,表明能量代谢系统崩溃;（4）MDA检测显示脂质过氧化水平较对照组升高4.3倍,荧光探针DCFH-DA证实胞内ROS累积量达1.2×10⁴RFU（较对照高8倍）,引发氧化应激级联反应;（5）SDS-PAGE和琼脂糖电泳显示,处理20分钟后胞内蛋白质条带强度衰减72%,基因组DNA出现明显弥散带,提示核酸-蛋白交联损伤。针对MRSA耐药性遗传基础,q PCR定量分析显示:EO处理对染色体定位的mecA基因和可移动遗传元件SCCmec均展现高效降解能力。在30 mM NaCl条件下处理20分钟,胞内mecA降解率99.98%,SCCmec基因降解率达99.93%。对体外游离ARGs（浓度1ng/μL）的降解实验进一步证实,EO可彻底消除基因片段（Ct值>35,低于检测限）。通过接合转移实验评估HGT风险发现:处理后的MRSA与受体菌的接合效率从初始4.3×10^-4降至未检出水平,表明EO通过破坏供体菌活性、降解游离质粒DNA双重机制,阻断了耐药基因的环境扩散。以青岛三甲医院二沉池出水（COD 272 mg/L,NH₃-N 4.09 mg/L,ARB浓度10⁴-10⁵CFU/mL）为对象进行中试验证。在5 mA/cm²电流密度下,20分钟内总菌落数从3.2×10⁴CFU/mL降至<1 CFU/mL（去除率>99.99%）。连续运行5000小时（208天）后,电极活性仅衰减7.5%,表明该电极较稳定,具有实际应用的基础。本研究表明,电化学氧化技术通过"活性氯氧化破膜-自由基损伤胞内组分-电场抑制基因转移"三重协同机制,实现了ARB与ARGs的同步去除。相较于传统工艺,其核心优势体现在:（1）无需外源消毒剂投加,避免致癌性DBPs生成;（2）对革兰阳性菌（如MRSA）灭活效率较氯化工艺提升2个数量级;（3）模块化设计适配分布式污水处理场景。未来通过电极材料优化与风光储一体化供电系统结合,有望为医疗废水、养殖废水等耐药性热点区域的污染控制提供绿色解决方案。更多还原

【Abstract】 The global proliferation of antibiotic resistance（AMR）has emerged as a critical public health challenge in the 21st century.According to World HealthOrganization（WHO）data,approximately 1.27 million deaths worldwide in 2019 were directly attributed to drug-resistant bacterial infections,with methicillin-resistant Staphylococcus aureus（MRSA）-notable for its multidrug resistance（to seven classes of antibiotics,includingβ-lactams and macrolides）and high mortality rate（20-40%for bloodstream infections）-ranking first among"superbugs."Conventional water treatment technologies such as chlorination and ultraviolet irradiation exhibit limited efficiency in removing antibiotic-resistant bacteria（ARBs）carrying resistance genes（ARGs）and may even accelerate horizontal gene transfer（HGT）by inducing DNA damage repair mechanisms.To address this challenge,this study innovatively developed a recirculating electrochemical oxidation（EO）system based on modified palladium-titanium electrodes,systematically investigating its dual efficacy in simultaneously removing the clinically critical ARB MRSA（strain ATCC43300）and its resistance gene cluster（SCCmec and mecA）,alongside elucidating the molecular mechanisms involved.A flow-through reactor（eelectrode area:9 cm²,electrode spacing:6 mm）was constructed using titanium cathodes modified with palladium nanoparticles via a joule-heating technique,paired with IrO₂/RuO₂-coated titanium anodes.SEM-EDS characterization revealed that the modified cathode formed a porous network structure with a 40%higher surface area than unmodified electrodes.XPS analysis confirmed the presence of PdO/PdO₂heterojunctions（binding energies:335.8 eV and 341.3 eV）,significantly enhancing chlorine evolution activity（Tafel slope reduced to 67.4 mV/dec）.In simulated medical wastewater（30 mM NaCl,pH7.4）,parameter optimization demonstrated that at a current density of 5 mA/cm²,rapid generation of active chlorine species（HClO/ClO^-）achieved 7-log bacterial inactivation（from 10⁷CFU/mL to below the detection limit）within 5 min.Reducing electrode spacing from 12mm to 6 mm increased reactive site density by 40%.Multiscale mechanistic studies revealed EO’s bactericidal pathways:（1）Transmission electron microscopy（TEM）showed 10–50 nm cell wall fractures and bilayer membrane disintegration after 3 min of treatment;（2）SYTO9/PI double staining confirmed membrane integrity loss in 99.7%of cells after 5 min;（3）Ion chromatography detected a 146%increase in intracellular potassium leakage（13 to 32 mg/L）,coupled with an 85%reduction in LDH enzyme activity（p<0.01）,indicating metabolic collapse;（4）MDA assays revealed a 4.3-fold elevation in lipid peroxidation,while DCFH-DA fluorescence probes quantified intracellular ROS accumulation at 1.2×10⁴RFU（8-fold higher than controls）;（5）SDS-PAGE and agarose electrophoresis demonstrated 72%attenuation in protein band intensity and diffuse genomic DNA bands after 20 min,suggesting nucleic acid-protein crosslinking damage.q PCR analysis demonstrated EO’s robust degradation capacity for MRSA’s genetic resistance determinants.Under 30 mM NaCl,20-minute treatment degraded 99.98%of intracellular mecA and 99.93%of SCCmec.Extracellular ARGs（1 ng/μL）were completely eliminated（Ct>35）.Conjugation experiments revealed that HGT frequency decreased from an initial 4.3×10^-4to undetectable levels,confirming EO’s dual mechanism of donor cell inactivation and plasmid DNA degradation.Pilot-scale validation using secondary effluent from a tertiary hospital in Qingdao（COD:272 mg/L,NH₃-N:4.09 mg/L,ARB:10⁴–10⁵CFU/mL）achieved>99.99%bacterial inactivation（3.2×10⁴to<1 CFU/mL）within 20 min at 5 mA/cm².Continuous operation for 5000 hours（208 days）resulted in only7.5%electrode activity decay,demonstrating exceptional stability for practical applications.This study establishes that electrochemical oxidation achieves simultaneous ARB and ARG removal through triple synergistic mechanisms:active chlorine-induced membrane disruption,radical-mediated intracellular damage,and electric field-inhibited gene transfer.Compared to conventional methods,its core advantages include:（1）Elimination of external disinfectant requirements,avoiding carcinogenic disinfection byproducts（DBPs）;（2）Two-order-of-magnitude higher inactivation efficiency for Gram-positive bacteria（e.g.,MRSA）than chlorination;（3）Modular design suitable for decentralized wastewater treatment.Future integration with optimized electrode materials and renewable energy systems holds promise for green remediation of AMR hotspots in medical and agricultural wastewater.更多还原