淮河磺胺类抗生素抗性基因污染特征及其扩散机制的研究

Due to the facts that antibiotics resistance genes(ARGs) could induce "superbugs" because of its widespread occurrence and rapid spread, so as to contribute menace to human health, modern instrument analytical method combined with molecular biological technique were used to develop a systematic study on sulfonamide resistance (SulR) genes in the typical area, Huaihe River watershed. A combination of field sampling and laboratory simulation was performed. To reveal the contamination characteristics of SulR genes, the occurrence and abundance of SulR genes were screened and characterized by common PCR and quantitative PCR in this area on the basis of determined the contamination of sulfonamides. Subsequently, the phylogeny, the carriers and the genetic structure of SulR genes were analyzed. The dissemination of SulR genes from sulfonamide-resistance strains to non-sulfonamide-resistance strain was futher investigated by filter mating, so as to reveal the dissemination mechanism of SulR genes. Results of this project will provide a rather scientific basis for the control of water pollution and the safety of drinking water in huaihe river and has important theoretical value and practical significance for improving the monitoring site of SulR and the establish of the safty evaluation and early warning system of the SulR in environment.

针对抗生素抗性基因（Antibiotic resistance genes, ARGs）广泛存在和迅速传播导致"超级细菌"出现对人类健康构成巨大威胁这一现状，本项目拟采用现代分析手段和分子生物学技术，野外采集和实验室研究相结合，对典型区域(淮河流域中游段)磺胺类抗生素抗性(Sulfonamide Resistance,SulR)基因污染开展系统研究。在明确该区域磺胺类抗生素污染的基础上，建立高灵敏度的普通PCR和荧光定量PCR检测技术，对SulR基因进行筛选和定量表征，揭示淮河SulR基因的污染特征。深入研究SulR基因的系统进化、向非抗性细菌的扩散条件及其内部遗传结构，揭示SulR基因连锁传播机制。项目研究成果为淮河流域水污染防治和确保饮用水安全提供科学依据，对完善我国SulR监测网点和建立SulR生态环境安全评价与预警体系具有重要的理论价值和实践意义。

抗生素抗性基因是一种新型环境污染物，其污染和传播给人畜健康带来了威胁。淮河是位于黄河与长江之间的一条重要河流，本项目对淮河中磺胺类抗生素（SAs）抗性基因的污染和传播开展了研究，取得了以下结果：.1 SAS抗性细菌在淮河水体和底泥中广泛存在，水体中的抗性细菌明显低于底泥，分别为1.16×104CFU/mL水和4.83×106CFU/g底泥；干流和支流水体中的抗性细菌相当；干流底泥中的抗性细菌高于支流。在耐药率方面，水体的耐药率为，干流和支流水体中的耐药率无显著区别，平均为16.44%；干流底泥的耐药率高于支流，平均为8.97%。.2 抗性细菌对SAs的敏感性存在差异，磺胺甲恶唑（SMZ）和磺胺嘧啶（SD）对各细菌的最小抑制浓度（MIC）范围分别为11-24 mg/mL和15-23 mg/mL。底泥中的抗性细菌对SMZ和SD的敏感性低于水体，MIC分别为19.47 mg/mL、19.67 mg/mL、17.00 mg/mL和16.76 mg/mL。.3 sul1和sul2基因是淮河中主要的SAs抗性基因，检出频率均为100%，其在水体和底泥中的丰度分别为1.21×109拷贝/L水、5.72×109拷贝/g底泥、3.32×108拷贝/L水和1.47×109拷贝/g底泥。干流底泥中sul1基因和sul2基因的丰度高于支流中相应基因的丰度；然而，干流水体中两者的丰度却低于支流相应基因的丰度。.4 整合酶基因int1在淮河中的检出频率为100%，其在水体和底泥中的丰度分别为2.38×109拷贝/ L水和2.29×109拷贝/g底泥。sul基因的丰度与int1基因显著正相关；然而，底泥中sul2基因丰度与int1基因却无显著相关性。.5 sul2基因是阴沟肠杆菌w-1对SAs产生耐药性的主要原因，该基因全长816bp，与链霉素耐药基因strA和strB形成sul2-strA-strB基因簇位于染色体上。其上游分别存在转座酶基因tnp 和整合酶基因int，其下游是一段反向重复序列，因此认为sul2基因主要通过转座子进行传播，虽然水平基因转移实验没有获得成功。总体上，本项目取得了预期的研究结果。