大肠埃希菌碳青霉烯异质性耐药及其分子机制研究

Heteroresistance, which has been demonstrated in antibiotic resistance phenomenon for several bacterial species to various catagories of antibiotics, is believed to be a prerequisite to full antimicrobial resistance. Due to the misleading false results from in vitro antimicrobial susceptibility tests, heteroresistance often results in clinical treatment failure. Therefore, research on heteroresistance will not only enhance the understanding of the evolution and the mechanisms of antibiotic resistance, but also provide accurate susceptibility information for the clinics. Our previous study has found Escherichia coli (E.coli)colonies with various sizes and morphologies in the inhibition zone of carbapenem by the K-B method, which were finially demonstrated to be heteroresistant isolates by population analysis profile (PAP). Authoritative researches has indicated that heteroresistance phenotype is due to the differential expression of antibiotic resistance genes of the same clone, which is associated with the duplication of antibiotic resistance genes and the alteration of genetic structures. Thus, we speculate that heteroresistance to carbapenem in Enterobacteriaceae is attributed to the differential expressions of the resistance determinants due to the duplication of the corresponding genes or the alteration of genetic structures.In order to elucidate the molecular mechnisms of carbapenem heteroresistance in E.coli and provide the right information for the clinics and slow down the emergence of full resistant strains, molecular biology techniques including PAP, PFGE, SDS-PAGE and FQ-PCR are adpoted to analyze not only the epidemiological characteristics and risk factors for carbapenem heteroresistance, but also the differential expression of the resistance genes and the mutations in porins genes and related regulators.

异质性耐药广泛存在于多种细菌对各类抗菌药物的耐药现象中，是细菌进展至完全耐药的必要前提。在药敏试验中常表现为假敏感而误导临床，引起治疗失败。因此，研究异质性耐药对了解细菌耐药过程及发生机制、为临床提供正确药敏信息具有重要意义。临床工作新发现：大肠埃希菌在碳青霉烯抑菌圈内有大小不等的菌落生长，PAP实验证实为碳青霉烯异质性耐药。权威杂志提出异质性耐药生长现象是同一克隆细菌耐药基因差异表达而形成的，与耐药基因扩增和遗传环境改变相关。由此推测：碳青霉烯耐药基因扩增或遗传环境改变所致耐药基因差异表达可能是大肠埃希菌碳青霉烯异质性耐药的主要机制。本项目拟采用PAP、PFGE、SDS-PAGE和FQ-PCR等方法，分析碳青霉烯异质性耐药大肠埃希菌的流行病学特征和危险因素，探讨耐药基因表达、膜孔蛋白及调控因子突变等分子机制，为阐明细菌耐药过程及机制、为临床提供正确药敏信息、减缓完全耐药菌出现提供新思路。

碳青霉烯类抗生素是治疗革兰阴性杆菌感染特别是产超广谱β内酰胺酶（ESBLs）和头孢菌素酶（AmpC）肠杆菌科细菌的最后一道防线。临床微生物工作中常常会发现大肠埃希菌碳青霉烯敏感菌株和耐药菌株共存的现象，即异质性耐药现象。由于异质性耐药株在体外环境中容易发生耐药表型的改变而表现为假敏感，不容易被识别，不容易被筛出，实验室不能给临床医师提供正确的药敏信息，常常导致临床治疗失败而延误病情。本研究以临床工作中发现的碳青霉烯异质性耐药大肠埃希菌为研究对象，通过流行病学调查、危险因子分析、PAP实验、PFGE、异质性耐药稳定性实验、时间-杀伤实验、FQ-PCR、SDS-PAGE和耐药基因遗传环境分析等实验确定大肠埃希菌对碳青霉烯异质性耐药的流行病学特征，并探讨其碳青霉烯异质性耐药的分子机制。本研究得出产ESBL酶为碳青霉烯异质性耐药的高风险因素，并且其菌株之间不存在爆发流行；证实了碳青霉烯异质性耐药的发生与ESBL酶高表达相关，并且与膜孔蛋白的缺失相关；同时证明了碳青霉烯异质性耐药菌株具有更强的毒力和致病性。因此，加强对大肠埃希菌碳青霉烯异质性耐药的认识和研究，对于全面了解细菌耐药过程及发生机制、为临床提供正确的药敏信息、优化抗菌药物使用策略、减缓完全耐药菌种的出现具有非常重要的现实意义。