【作者】 李娜；

【导师】 何朝珠；

【作者基本信息】 南昌大学 ， 护理学， 2025， 博士

【摘要】 研究背景:口腔综合治疗台水路（Dental Unit Waterlines,DUWLs）内生物膜的定植,是口腔科医院交叉感染的重大风险源之一。DUWLs特有的管腔结构与间歇性水流特征,导致管腔内出现层流现象,为细菌的粘附提供了理想的环境。由此导致DUWLs微生物污染率高达58.59%。木糖葡萄球菌和蜡样芽孢杆菌等易形成顽固性生物膜的条件致病菌定植率高,这些细菌会显著提升口腔科交叉感染事件发生的概率,严重威胁临床安全。现行的化学消毒方案虽能实现即时抑制浮游菌的效果,但存在持续消毒导致管路腐蚀、医护人员职业暴露风险及环境污染加剧等危害,且消毒失当将直接诱发生物膜再次定植的根本性矛盾。因此,探索具备生物膜清除、低腐蚀性及环境友好特性的新型消毒剂,是实现口腔科医院感染控制的重要途径。研究目的:本研究通过多中心横断面调查,了解某市不同级别医疗机构DUWLs污染的现状,筛选出形成生物膜能力强的菌种。在此基础上,对比评价绿原酸与含氯消毒剂对DUWLs生物膜的抑制效能、管路设备腐蚀及消毒副产物生成的影响;进一步通过RNA-seq、扫描电镜及共聚焦等技术,探究绿原酸抑制微生物生物膜形成的作用机制,旨在为制定口腔诊疗感染防控策略提供理论依据和实践方向。研究方法:1.DUWLs细菌培养及菌种的筛选采用分层随机抽样法,采集本地区不同级别医疗机构口腔综合治疗台（Dental Chair Unit,DCU）的水源水、手机水、三用枪水和漱口水。采集的样本涂布于营养琼脂平皿和军团菌琼脂平皿,分别置入恒温培养箱和二氧化碳培养箱进行培养。选用全自动菌落计数仪进行活菌计数,并结合Sanger测序和MALDI--TOF飞行时间质谱,解析培养方法获得的微生物群落构成。此外,采用结晶紫染色法对采集菌种的生物膜形成进行定量分析,筛选出形成生物膜能力强的菌种。2.绿原酸与含氯消毒剂对生物膜抑制效能、管路设备腐蚀及消毒副产物生成的影响研究采用微量肉汤稀释法测定绿原酸对易产生生物膜的标准菌株--表皮葡萄球菌（ATCC35984）和鲍曼不动杆菌（ATCC17978）的最小抑菌浓度。采用结晶紫染色法,比较最小抑菌浓度下的绿原酸和含氯消毒剂对分离得到的微生物生物膜形成的抑制作用。通过扫描电镜和X射线光电子能谱,比较绿原酸、含氯消毒剂对DCU电磁阀的金属元件和管道的腐蚀程度。通过大气采集器和液相色谱等分析方法,比较DCU操作单元的空气中以及消毒前后水样中各类副产物的含量。3.不同消毒剂干预后微生物群落和生物膜差异的分析采用16s rDNA扩增子测序的方法,利用生成的操作分类单元信息,并从纲、门、科、属、种的水平,分析不同消毒剂（含氯消毒剂、绿原酸）和消毒前后微生物群落构成和分布丰度差异;采用扫描电镜观察不同消毒剂干预前后DUWLs生物膜含量的变化。4.绿原酸抑制细菌生物膜的机制研究采用扫描电镜和激光共聚焦显微镜,比对绿原酸、含氯消毒剂和蒸馏水对高成膜微生物的生物膜微结构及膜内细菌活性差异;通过RNA-seq的方法,比较绿原酸作用于本研究中生物膜形成量最多的两种菌--蜡样芽胞杆菌和木糖葡萄球菌后的转录组差异,初步阐明新型DUWLs消毒剂--绿原酸的生物膜清除机制。研究结果:1.某市DUWLs污染现状及菌群生物膜特征共调查55家医疗机构,214台DCU。在2,782份水样中,除时段4外,余时段均为手机水的合格率最低。各时段中未进行排水的水样菌落中位数最高（手机水:1,240 CFU/m L,合格率10.75%）。定期消毒能显著降低各出水口菌落数（OR=4.767,P<0.001）。除此之外,此次调查显示,仅23.64%的机构定期消毒DUWLs（13/55）,其中76.92%的机构采用含氯消毒剂进行消毒（10/13）。在分离培养获得的60种2,123株分离菌株中,蜡样芽胞杆菌和木糖葡萄球菌等8株菌展现强生物膜形成能力。2.绿原酸与含氯消毒剂对生物膜抑制效能、设备腐蚀及消毒副产物生成的影响绿原酸和含氯消毒剂两种消毒剂均能抑制各类生物膜的形成,且绿原酸对蜡样芽胞杆菌、铜绿假单胞菌和偶发贪铜菌生物膜抑制效能显著优于含氯消毒剂。进一步的研究发现,含氯消毒剂组处理的电磁阀金属元件和聚氨酯管路均出现结构断裂,表面元素变化和腐蚀严重程度显著高于绿原酸组。此外,含氯消毒剂消毒时空气中、消毒后水样中三氯甲烷和二氯乙酸等消毒副产物浓度高于绿原酸。3.不同消毒剂干预对DUWLs菌群动态变化及生物膜结构的影响三组菌落数呈现差异变化:蒸馏水组干预第1周菌落数最高（7,500CFU/m L）,且持续保持高污染状态（1--6周合格率<35%）;含氯消毒剂组在干预4周后菌落数显著下降（第8周达300 CFU/m L,合格率稳定在90%以上）;绿原酸组虽初期波动较大（4--8周合格率50%--90%）,但9周后维持100%达标。扫描电镜结果显示,绿原酸通过破坏生物膜三维基质增强杀菌渗透性,且绿原酸干预3个月后残留生物膜的量低于含氯消毒剂。绿原酸组和含氯消毒剂组16s rDNA扩增子测序结果显示,属水平上,二者整体细菌群落组成相似,均以新鞘氨醇杆菌属等为优势菌属,且与蒸馏水组比较,绿原酸组和含氯消毒剂组军团菌属和分支菌属相对丰度更低。门水平上,三组均以变形菌门为优势菌门,且共含拟杆菌门、疣微菌门等;不论是门水平还是属水平,绿原酸组微生物群落的丰富度远高于含氯消毒剂组。4.绿原酸抑制生物膜形成的作用机制解析绿原酸和含氯消毒剂处理均显著降低了8种高生物膜形成能力的菌株的生物膜量及活菌载量。进一步的RNA-seq分析显示:绿原酸显著下调木糖葡萄球菌his B/his C（咪唑甘油磷酸脱水酶,Log₂Fold Change=-2.47/2.57）和蜡样芽胞杆菌gap B（甘油醛-3-磷酸脱氢酶,Log₂Fold Change=-6.75）等的表达。KEGG富集分析显示,绿原酸干预显著抑制蜡样芽胞杆菌和木糖葡萄球菌的代谢途径（A09100,P<0.05）,其中,蜡样芽胞杆菌的氨基酸代谢途径受抑制的基因数量最多为22个,而木糖葡萄球菌的碳代谢途径受抑制的基因数量最多为17个。研究结论:1.本研究分离获得的60种2,123株分离菌株中,蜡样芽胞杆菌和木糖葡萄球菌等8种菌展现强生物膜形成能力,增加了口腔科医院感染的机会。2.绿原酸消毒剂抑制多种细菌生物膜的形成效果优于含氯消毒剂,且对DUWLs重要元件的腐蚀及副产物生成的危害低于含氯消毒剂,从多维度展现出优势。3.绿原酸与含氯消毒剂均能有效降低DUWLs中的菌落数并提升合格率,但绿原酸呈现出多元化的抑菌机制,导致其组内微生物群落的丰富度远高于含氯消毒剂组,并能显著抑制蜡样芽胞杆菌和木糖葡萄球菌生物膜形成的代谢途径,这些发现为其在医院感染控制的应用,提供了分子机制层面的理论支撑。更多还原

【Abstract】 BackgroundThe colonization of biofilms within dental unit waterlines（DUWLs）poses a significant risk of cross-infection in hospital dental departments.The unique lumen structure and intermittent water flow in DUWLs create laminar flow phenomena within the tubing,providing an ideal environment for bacterial adhesion.This results in a microbial contamination rate as high as 58.59%in DUWLs,with a high colonization rate of opportunistic pathogens such as Staphylococcus xylosus and Bacillus cereus,which are prone to forming resilient biofilms.These bacteria substantially increase the likelihood of cross-infection events,severely compromising clinical safety.While current chemical disinfection protocols effectively inhibit planktonic bacteria,they induce secondary risks such as material corrosion from prolonged use,occupational exposure for healthcare workers,and environmental pollution.Moreover,improper disinfection practices may directly trigger biofilm re-colonization.This highlights the urgent need to explore novel disinfectants with biofilm removal capabilities,low corrosivity,and environmental friendliness,offering innovative solutions for nosocomial infection control in dentistry.Objectives1.Investigate microbial contamination in DUWLs across healthcare institutions of varying tiers through cross-sectional sampling,identifying biofilm-forming bacterial strains.2.Evaluate the efficacy of chlorogenic acid（CGA）and chlorine-based disinfectants in inhibiting DUWLs biofilms,assessing their impacts on equipment corrosion and disinfection byproducts.3.Elucidate the mechanism by which CGA suppresses microbial biofilm formation,providing theoretical and practical insights for optimizing infection control strategies in dental care.Methods1.DUWLs Microbial Cultivation and Strain SelectionSamples（source water,handpiece water,air/water syringe water,and mouth rinse water）were collected from dental chair units（DCU）across different-tier medical institutions via stratified random sampling.Samples were plated on nutrient agar and*Legionella*agar,incubated under controlled conditions.Automated colony counters quantified viable bacteria,while Sanger sequencing and MALDI--TOF mass spectrometry characterized microbial communities.Crystal violet staining quantified biofilm formation,identifying high-potential biofilm-forming strains.2.Comparative Analysis of CGA and Chlorine-Based DisinfectantsMicrobroth dilution assays determined CGA’s minimum inhibitory concentration（MIC）against biofilm-forming reference strains（Staphylococcus epidermidis ATCC35984 and Acinetobacter baumannii ATCC17978）.Biofilm inhibition at MIC levels for CGA and chlorine-based disinfectants was assessed via crystal violet staining.Scanning electron microscopy（SEM）and X-ray Photoelectron Spectroscopy（XPS）evaluated corrosion on DCU solenoid valves and tubing.Airborne and aqueous disinfection byproducts were quantified using high-volume air samplers and high-performance liquid chromatography.3.Microbial Community Dynamics Post-Disinfectant Intervention16S rDNA amplicon sequencing analyzed shifts in microbial composition at Class,phylum,family,genus,and species levels across disinfectant-treated groups（chlorine-based,CGA）and untreated controls.SEM visualized biofilm structural alterations pre-and post-treatment.4.Mechanistic Study of CGA-Mediated Biofilm DisruptionSEM and confocal laser scanning microscope（CLSM）compared biofilm microstructure and bacterial viability in CGA-versus chlorine-treated groups.RNA-seq analysis identified transcriptomic differences in Bacillus cereus and Staphylococcus xylosus following CGA exposure.KEGG pathway enrichment clarified metabolic targets of CGA action.Results1.DUWLs Contamination Status and Biofilm CharacteristicsOf 55 institutions（214 DCU）surveyed,2,782 water samples revealed handpiece water as the most contaminated（median colony count:1,240 CFU/m L;compliance rate:10.75%）.Routine disinfection significantly reduced bacterial loads（OR=4.767,p<0.001）.Only 23.64%of institutions performed regular DUWLs disinfection（76.92%using chlorine-based agents）.Among 2,123 isolates from 60 taxa,8strains—including Bacillus cereus and Staphylococcus xylosus—exhibited robust biofilm-forming capacity.2.Efficacy of CGA versus Chlorine-Based DisinfectantsBoth agents inhibited biofilm formation,with CGA outperforming chlorine against Bacillus cereus,Pseudomonas aeruginosa,and Cupriavidus pauculus.Both solenoid metal components and polyurethane tubing treated with chlorine groups showed structural fractures,with surface elemental changes and corrosion severity significantly higher than those in the CGA groups.Chlorine disinfection generated higher levels of trihalomethanes and dichloroacetic acid in air and water samples.3.Dynamic Shifts in Microbial Communities Post-TreatmentThree distinct trends emerged:Untreated controls maintained high contamination（7,500 CFU/m L;<35%compliance）.Chlorine groups achieved>90%compliance by Week 8 but required frequent reapplication.CGA groups stabilized at 100%compliance by Week 9 with no rebound.SEM revealed CGA disrupted biofilm 3D matrices,enhancing bactericidal penetration.16S sequencing showed higher microbial diversity in CGA groups（Proteobacteria-dominant）,both CGA groups and chlorine groups with reduced Legionella and Mycobacterium abundance versus Untreated controls.4.Mechanistic Insights into CGA-Driven Biofilm SuppressionCGA reduced biofilm biomass and viable cell counts more effectively than chlorine.Transcriptomic analysis identified significant downregulation of his B/his C（imidazole glycerol phosphate dehydratase,Log₂FC=-2.47/2.57）in Staphylococcus xylosus and gap B（glyceraldehyde-3-phosphate dehydrogenase,Log₂FC=-6.75）in Bacillus cereus.KEGG enrichment confirmed CGA disrupted amino acid（22 genes）and carbon metabolism（17 genes）pathways in these species.Conclusions1.Eight high-risk biofilm-forming strains—predominantly Bacillus cereus and Staphylococcus xylosus—were identified among 2,123 isolates,heightening nosocomial infection risks.2.CGA surpasses chlorine-based disinfectants in biofilm inhibition,material compatibility,and byproduct safety,demonstrating superior potential for DUWLs disinfection.3.CGA ensures sustained microbial control with higher ecological diversity preservation and targeted suppression of pathogenic metabolic pathways,positioning it as a promising agent for clinical infection prevention in dental settings.更多还原