水源水中低浓度红霉素药物的共代谢机制及对其抗药性细菌的选择性效应

The residue of antibiotics, as well as the rapid evolution and dissemination of antibiotic resistant bacteria in the environmental waters have posed new challenges to the source water treatment. The efficiency of source water treatment process is the key factor for controlling the entry of these micro-pollutants to drinking water. In this work, the biological aerated filter (BAF) will be set up to study the biodegradation and biotransformation mechanism of the erythromycin (ERY), which is one of the typical antibiotics presenting in the source water, and the influence of environmental factors on the co-metabolism of ERY will be explored. Furthermore, the effect and toxicity of ERY and its biodegradation products on the functional microbial community during the BAF process will be investigated, and structural characteristics of the predominant microbial community under the selective pressure of persistent low-concentration ERY and its biodegradation products will be also identified. In addition, the induction mechanism of biofilm formation for the functional bacteria, and the influence on the evolution of antibiotic resistant determinants by ERY and its biodegradation products will be both discussed. The protection and release mechanisms of survival and propagation of these antibiotic resistant determinants by the biofilm will be studied. The correlation of ERY and its biodegradation products with corresponding resistant determinants both in water and biofilm phases will also be investigated. Finally, the gene-based biofilm resistance mechanism under the persistent selective pressure of low-concentration ERY and its biodegradation products will be clarified. Findings of this study will provide significant information for the drinking water quality security and enrich the scientific theory of the control techniques for emerging contaminants in source water.

微量抗生素药物在环境水体中的残留和抗药性菌群的快速演变与散播对水源水处理是一新的挑战，水源水处理工艺的有效性是控制这些微污染物进入饮用水中的关键。建立水源水预处理装置-曝气生物滤池，研究典型抗生素红霉素（ERY）在曝气生物滤池处理过程中的生物降解转化机制，探明相应环境生态因子对ERY共代谢的影响机制；考察ERY及其降解产物对功能微生物群落结构的演变影响和毒理学效应，识别在持久性低浓度ERY及其降解产物的选择性压力下优势微生物群落的结构特征；探讨ERY及其降解产物对功能细菌生物膜形成的诱导机制，以及对降解微生物抗药性因子的演变影响，研究生物膜对抗药性因子生存和繁殖的保护及释放机制，掌握ERY及其降解产物与抗药性因子之间的相关关系，在基因层面上阐明生物膜在低浓度ERY及其降解产物选择性压力下的抗药性机制。项目对于保障饮用水水质安全，丰富水源水中新兴微污染物控制技术的科学理论具有重要的现实意义。

细菌抗药性问题已成为全球环境生态安全及人体健康领域的一个关注焦点和研究热点。目前关于环境中的抗生素对细菌抗药性的产生和传播作用机制还尚不清楚。美国环保署于2009年首次将红霉素列为饮用水标准需优先检测和控制的候选污染物之一，这无疑给水源水处理提出了新的要求和挑战。本项目以红霉素（ERY）作为研究对象，建立了水源水中微量ERY的SPE-HPLC-MS/MS检测方法，及红霉素抗性基因（ERY-ARGs）的qPCR检测方法；通过梯式驯化方法，筛选分离获得ERY高效降解菌株ERY-E，并成功将其接种于曝气生物滤池（BAF）生物强化去除ERY；系统研究了ERY的生物降解特性和共代谢降解机制，分析了其降解动力学过程，并对其共代谢降解酶系的分离及活力表征进行初步探讨；考察了ERY降解微生物群落在ERY、TCS和重金属共同暴露下的抗药性演变机制，探讨了这些选择性因子与ERY-ARGs之间的相关关系。结果表明，所建立的SPE-HPLC-MS/MS方法对水源水中ERY的检出限可达4.5ng/L，定量限为14.9ng/L；ERY-ARGs qPCR方法扩增效率在84~102%之间。筛分获得的菌株ERY-E经16S rDNA测序鉴定为假单胞菌属，当体系温度为30ºC和pH为7.0时，其对ERY的去除率可达65.4%，当加入10mg/L的葡萄糖、牛肉膏和酵母粉时，可对ERY去除率分别提高8.4%、12.9% 和18.3%，表明菌株对ERY的降解属于共代谢机制。通过对ERY降解过程进行动力学拟合，发现其符合一级反应动力学特征。将菌株ERY-E接种于BAF，发现低浓度ERY对水源水处理优势微生物群落的选择性作用较小，且菌株ERY-E可与生物膜固有微生物协同生存，对ERY的去除率可达60.6%，而且BAF对ERY去除率随着HRT和A/L的增大而递增。由相关性分析可知，ERY和TCS与某些ERY-ARGs [如mef(A)/mef(E)和ere(A)]之间具有显著相关性，表明TCS可能对ERY-ARGs具有交叉选择性作用；重金属Zn和Pb与某些ERY-ARGs [如mef(A)/mef(E)和ere(B)]之间同样具有显著相关性，表明这些重金属对ERY-ARGs具有共选择效应。本项目研究成果可为水源水中微量难降解有机污染物的有效去除，及饮用水安全保障提供了新的可行性思路，具有重要现实意义和研究价值。