海水浴场肠杆菌科细菌耐药影响机制及抗性基因转移研究

The detection rate of antibiotic resistance Enterobacteriaceae is high in marine environment. It would be a great threat for the public with the spread of bacteria and resistant genes. In particularly, there are trends of the spread in floating population for "Super resistance gene" NDM-1 of carbapenemase resistant Enterobacteriaceae.Three bathing beaches were selected for study on the resistant characteristic from the perspectives of time and space; The drug sensitivity test,Realtime-PCR, plasmid transformation and bacterial conjugation experiments will be used to establish the multi-level approach for the study on influencing mechanism ,which will detect it from bacterial communities , antibiotic resistance genes , mRNA and expression of the enzyme.We attempt to clarify the prevalence of ESBLs,CRE,and dominant bacterium on beaches and the ability of transfer of ARGS in different environment.The feces will be collected from the high risk population and the control group to investigate the possible spread of ARGS between the beaches and populations, and the homologous analysis of bacterium will be analyzed by ERIC-PCR. The human sourced Enterobacteriaceae which have high homology with environment,the Enterobacteriaceae with dangerous ARGS will be cultured and the environmental risk will be predicted.It is innovative that the mechanism of resistance were explored from the UV rays, from organics and other environmental factors, blaNDM genes and bacteria of the populations which were investigated.The study can provide theoretical and practical basis for avoiding the risk of population transmission of antibiotic-resistant bacteria and ARGS on bathing beaches, and provide basic data for the research of antibiotic resistance Enterobacteriaceae and populations on the bathing beaches.

海洋肠杆菌科细菌检出率较高，该菌及抗性基因传播对公众有很大健康威胁。其中"超级耐药基因"NDM-1能随人群迁移而快速传播。本研究对3个海水浴场采样，筛选肠杆菌并检测环境因子。通过细菌培养、药敏实验、RealtimePCR、基因及酶检测，建立基于种群、基因、mRNA及酶水平的多层级方法,考察耐药菌及抗性基因的时空变化规律及环境影响机制。明确优势耐药菌、ESBLs菌、耐碳青霉烯肠杆菌及抗性基因等流行状况；探讨环境对抗性基因转移的影响。对人群粪便细菌进行转化及接合实验、ERIC-PCR方法分析环境及人群肠杆菌的同源性及抗性基因的传播。培养多重耐药菌，评估其环境风险。该研究前期建立了成熟的环境检测及抗性基因研究方法。从紫外线、富营养因子等角度探讨耐药影响机制；进行blaNDM及人群研究；具有较好的创新性。该研究可为规避浴场耐药菌的人群风险提供理论及实际依据,为抗性基因的环境及人群研究提供基础资料。

水环境是耐药基因的储存库，海水浴场人群可能因此面临健康风险。. 本研究筛选浴场细菌267株，肠杆菌的优势菌为肺炎克雷伯菌。主要菌株为：肺炎克雷伯、阴沟肠杆菌、产气肠杆菌。其耐药较高的抗生素为：氨苄西林（AMP）、二代头孢、氨苄西林/舒巴坦、复方新诺明(SXT)等；枯水期菌株的种类较丰水期少。. FOX(头孢西丁)在两浴场的菌株耐药率差异有统计学意义，水中耐药率较沙低（AMP、FOX、TIM）。.浴场检出tetA、qrns、Sul1、qep、qrns、intI、DHA、可移动元件ISCR1等。其中int1、tetA、sul-1较多。intI主要为aadA1及aadA2。.基于mcr-1的风险，本研究发现4株mcr-1阳性菌株，1株人体来源为大肠杆菌，3株环境来源为假单胞菌、不动杆菌及大肠埃希氏菌。.对N、P、COD、UV254与耐药菌进行相关分析，只有铵盐与菌株的耐药差异有统计意义。.亚抑菌浓度抗生素培养菌株SYW62的intI突变分析表明：头孢他啶培养时突变较氧氟沙星大，突变位点在260-272之间，主要为Cys插入及缺失。.278株浴场从业人群细菌主要为大肠杆菌、少量阴沟肠杆菌及不动杆菌，其ESBLs菌主要基因型为CTX-M-14及CTX-M-27。氨苄西林耐药率在环境及人源菌中均最高，随后为复方新诺明、氯霉素及二代头孢，从业人群与环境菌耐药高度相似。. 以EC600为受体菌，肺炎克雷伯为供体菌，接合成intI 率为50%，其中28和71a接合效率高；低剂量紫外线和盐度对菌生物量有影响，耐药影响有限。以tetA及mcr-1为目的基因，realtimePCR表明其表达趋势与生物量一致。. 细菌ERIC及PFGE同源性分析表明，菌株B37和B71A相似度为100%；B22、B34、B52、B53的相似度均为100%，该4株菌分别来自大东海和三亚湾的沙样，极可能通过人群活动而传播；B37和B71A分别来自三亚湾和亚龙湾的沙样和水样。从业人群的22株ESBLs大肠杆菌中DDH73B、DDH65、DDH53b、DDH39a属于同一个带型，流行病学具有高度关联。.研究提示复方新诺明、多粘菌素等应在医疗和养殖中严加管理，减少风险。浴场沙滩对人群具有一定的风险，小剂量盐度和短期紫外的暴露对耐药影响较小，转录的差异到耐药酶表达可能存在滞后或受非编码RNA影响，其机制需要进一步探讨。