不动杆菌新blaOXA同源基因鉴定、功能及转移机制研究

Acinetobacter spp. has emerged as one of the most important opportunist pathogens and spread widely across China, especially the carbapenem-resistant isolates. Carbapenem-hydrolysing OXA enzymes were considered as the most significant determinants of carbapenem resistance in this species. It has been confirmed that transferable elements, such as transposons, which were involved in the transmission of blaOXA genes among different species, and contributed to the prompt increase of carbapeneme-resistant rates. Our previous study revealed that the homologous genes of OXA enzymes could be identified in Acinetobacter venetianus, which can get into Acinetobacter baumannii by horizontal gene transfer and cause carbapenem resistance. This study will dedicate to identify novel blaOXA homologous genes, demonstrate the genetic location, genetic structures and potential dissemination ability by high-throughput genome sequencing technology and bioinformatics approach. We will also clarify the carbapenem hydrolysis activity of OXA enzymes by gene expression and enzyme kinetics to describe the roles of blaOXA genes in Acinetobacter identification and antibiotic resistance mechanism. Overall, we hope to find the prevention, control strategy and also search for new antibiotic target sites for this clinical pathogen.

不动杆菌属细菌临床分离率不断上升，已成为医院感染最常见的病原菌之一，尤其是碳青霉烯类抗生素耐药鲍曼不动杆菌已在我国广泛流行，具有碳青霉烯水解活性的OXA酶是介导其耐药的最重要机制。现有研究表明，blaOXA基因可通过转座子等移动元件在不动杆菌属各菌种间传播，从而导致其对碳青霉烯类抗生素耐药率迅速上升。我们前期研究已在威尼斯不动杆菌（A.venetianus）中发现了OXA型碳青霉烯酶同源基因，该基因极可能通过基因水平转移等方式进入鲍曼不动杆菌，造成碳青霉烯类抗生素耐药。本研究将应用高通量基因组测序技术及生物信息学方法，鉴定不动杆菌新blaOXA同源基因，明确其定位、周围结构及潜在的播散能力。并通过基因表达及酶动力学研究，明确新OXA酶碳青霉烯类抗生素的水解活性。为系统阐述blaOXA基因在不动杆菌属菌种鉴定及耐药中作用，制定合理的不动杆菌防控措施及寻找新型抗菌药物靶位点奠定基础。

碳青霉烯耐药鲍曼不动杆菌在我国广泛流行，给临床抗感染治疗与院感防控工作带来严峻挑战，具有碳青霉烯水解活性的OXA酶是介导鲍曼不动杆菌对该类抗生素耐药的主要机制。本研究通过Southern杂交、质粒接合转化与酶切克隆测序等方法明确我国鲍曼不动杆菌blaOXA-23基因的定位及携带其传播的可移动元件；对携带blaOXA-23基因的质粒进行全质粒测序，分析耐药质粒特征；对染色体携带blaOXA-23基因的菌株通过qRT-PCR测定其表达量，并应用PacBio RS II第三代高通量测序技术，对随机挑选的5株碳青霉烯耐药鲍曼不动杆菌进行基因组完成图测定，以阐明blaOXA-23基因倍增与其耐药性关联的分子机制，从而系统揭示我国鲍曼不动杆菌blaOXA-23基因的传播机制。研究发现，克隆复合体CC92是我国分布范围最广的携带blaOXA-23基因的碳青霉烯耐药鲍曼不动杆菌流行克隆，blaOXA-23基因可定位于染色体或质粒上。CC92克隆的垂直传播与可移动元件（可接合质粒pAZJ221与转座子Tn2009）的水平传播是导致blaOXA-23基因在我国鲍曼不动杆菌中广泛流行的主要原因。本研究还进一步明确了碳青霉烯耐药鲍曼不动杆菌blaOXA-23基因倍增与其耐药性的关联，即尽管有大约38%的菌株携带2个或者更多拷贝的blaOXA-23基因，但blaOXA-23基因的拷贝数与亚胺培南的最小抑菌浓度（MIC）之间并无明显相关性，blaOXA-23基因多拷贝并不能显著增强碳青霉烯耐药鲍曼不动杆菌对亚胺培南的耐药性。blaOXA-23基因多拷贝可通过串联性多片段聚集或独立的遗传事件随机插入到鲍曼不动杆菌基因组序列中。而携带单拷贝blaOXA-23基因的碳青霉烯耐药鲍曼不动杆菌经体外抗生素诱导后，菌株对亚胺培南MIC、blaOXA-23基因表达量及拷贝数均存在明显上升趋势。提示除blaOXA-23基因表达量及拷贝数两方面因素外，可能还存在其他未知因素影响鲍曼不动杆菌对碳青霉烯类抗生素耐药，需要进一步深入探索。综上所述，blaOXA-23基因通过耐药克隆的垂直传播及质粒、转座子等可移动元件的水平传播，是造成我国鲍曼不动杆菌对碳青霉烯类抗生素耐药的主要原因。本研究可为阐明鲍曼不动杆菌的传播机制开辟新思路，也为今后制定合理的流行病学监测与防控措施提供科学依据。