我国阿奇霉素和三代头孢类抗生素共耐药志贺菌的流行特征与传播进化规律研究

Shigellosis, caused by Shigella genus remains an important public health threat globally. Emerging multidrug-resistant (MDR) isolates have exacerbated the global burden, leading to increased morbidity, mortality, and treatment costs. During the routine surveillance of shigellosis, we identified the emergence and prevalence of MDR isolates with concurrent resistance to azithromycin and third-generation cephalosporins in China, which had even caused a waterborne outbreak of shigellosis. So the potential threat to public health from the changes in prevalence and antimicrobial resistance of Shigella species with resistance to azithromycin and third-generation cephalosporins in Mainland China should not be underestimated. Here we will conduct the study on the epidemiological characteristics and changing trend of the Shigella isolates with resistance to azithromycin and third-generation cephalosporins in Mainland China. Furthermore, we will explore their molecular mechanisms conferring MDR especially resistance to azithromycin and third-generation cephalosporins by using antimicrobial susceptibility testing, resistance genes screening, plasmid conjugation transfer, southern blotting and plasmid sequencing. Then we will select the representative Shigella strains with resistance to azithromycin and third-generation cephalosporins and submit for whole genome sequencing to clarify the role of transposons, insertion/deletion, and plasmids in the development, spread, variation, and evolution of antimicrobial resistance. Additionally, phylogenetic analysis based on single-nucleotide polymorphisms and core-pan genome was performed to elucidate the evolution and dissemination of Shigella isolates with resistance to azithromycin and third-generation cephalosporins in China. In conclusion, our present study can provide the scientific basis for the shigellosis surveillance, early warning and confirmation of the outbreaks, source-tracking, transmission survey, and formulation of the control and prevention strategies.

志贺菌引起的细菌性痢疾仍然是一个严重危害公共卫生健康的全球性问题，特别是多重耐药菌株的出现与流行给公共卫生安全带来了更大威胁。本课题组在细菌性痢疾的监测中发现了耐阿奇霉素和三代头孢类抗生素的多重耐药志贺菌的出现与流行，并首次发现了一起水源性菌痢暴发疫情。志贺菌这种罕见耐药表型及其对公共卫生安全的威胁不容忽视。本项目拟在前期工作基础上，深入系统地开展阿奇霉素和三代头孢类抗生素共耐药志贺菌的流行病学监测，明确其在我国的流行特征与变异变迁规律；通过抗生素敏感性试验、耐药基因检测、接合转移、southern杂交、质粒测序等分析，揭示志贺菌对阿奇霉素和三代头孢类抗生素共耐药的分子基础，通过代表性菌株的全基因组测序和生物信息学分析，阐明转座子、质粒等遗传元件在耐药性产生、传播与进化中的作用；通过系统发生分析与种群结构重建，探究共耐药志贺菌在我国的传播演化规律，为我国细菌性痢疾的治疗和防控提供科学依据。

志贺菌引起的细菌性痢疾仍然是一个严重危害公共卫生健康的全球性问题，特别是多重耐药菌株的出现与流行给公共卫生安全带来了更大威胁。本项目深入系统开展了我国阿奇霉素和三代头孢类抗生素共耐药志贺菌的流行特征与传播进化规律研究。通过对实验室分离鉴定的4237株志贺菌进行抗生素敏感性试验，筛查出1327（31.3%）株对头孢曲松耐药的志贺菌，筛查出头孢曲松和阿奇霉素共同耐药志贺菌399株，总检出率为9.4%；而且随着时间的发展，宋内和福氏志贺菌中共同耐药志贺菌的检出率在不断上升。共同耐药志贺菌的耐药结果显示，耐药水平最高的抗生素是替卡西林（99.7%）、氨苄西林（99.5%）和头孢唑啉（99.5%）。耐药基因检测结果显示共同耐药志贺菌均含有介导阿奇霉素耐药的mph(A)基因，以及介导头孢类耐药的ESBLs耐药基因。通过对携带 blaCTX-M和mph(A) 基因的共同耐药志贺菌进行S1-PFGE和Southern杂交分析，结果显示blaCTX-M和mph(A) 基因主要定位在三种质粒类型上，其大小分别为70kb、100kb和135kb左右。我们对20株质粒接合成功的阳性接合子进行了药敏试验，结果显示均对头孢曲松和阿奇霉素耐药。通过对携带blaCTX-M和mph(A)基因的志贺菌进行质粒提取及测序分析，结果显示10株共同耐药志贺菌中有8株为IncF型质粒，1株IncI型质粒和1株IncB/O/K/Z型质粒。而且发现共耐药志贺菌质粒上的mph(A)基因上下游分别都有IS26和IS6100的存在；而在blaCTX-M基因的上游都有ISEcp1。分子钟的相关分析显示，共耐药菌株的共同祖先出现时间约为1983年，另外暴发菌株作为共耐药菌株的重要组成部分，其共同祖先的出现时间约在2010年。以上研究结果为细菌性痢疾的耐药监测、暴发疫情的识别、传染源追溯、传播关系的调查以及防控策略的制订提供了科学依据。