基于噬菌体核酸检测的病原菌及其耐药性快速检测新方法

Rapid identification and antibiotic susceptibility testing of pathogenic bacteria would be of great significance to give appropriate treatments and control of the spread of drug-resistant bacteria. Traditional methods in the laboratory for identification and drug resistance detection of pathogens are based on phenotype, which are labor-intensive and time-consuming. Molecular diagnosis assays are rapid but suitable only for the known mutations conferring resistance. Based on the detection of phage amplification by quantitative PCR, the applicant had established a rapid detection system for Acinetobacter baumannii in serum samples. The aims of the present study are to develop new rapid drug resistance detection methods based on developed system, and to develop new systems for rapid identification of pathogenic bacteria and drug susceptibility testing through phage nucleic acid detection. For achieving the objectives of this project, we will screen bacteriophages with broad spectrum and high bactercidal efficiency against Staphylococcus aureus, Acinetobacter baumannii and Mycobacterium tuberculosis. Based on genome sequencing and bioinformatics results of the newly-isolated phages, specific PCR primers and probes would be designed. The developed assays are based on the specificity of the phages and exponential bursts during the phage replications to detect the host bacteria through monitoring the changes of the phage nucleic acid by qPCR or droplet digital PCR. Since phages propagate only through viable bacteria, the rapid antibiotic susceptibility testing methods would be developed based on above systems according to the change of the phage copies with and without the additions of the antibiotics. This project will build a new rapid identification method of bacteria and drug resistance, and would have significant social benefits and bright market prospects.

病原菌及其耐药性的快速检测对于病人诊疗、控制耐药菌株的传播有着重要意义。基于表型检测的传统方法，耗时长且工作量大。分子检测方法能快速给出结果，但耐药性的检测局限于已知的耐药机制。申请人前期建立了一种基于荧光定量PCR检测噬菌体扩增的快速检测血清样本中鲍曼不动杆菌的体系。本课题进一步探索将其用于快速药敏检测，发展基于噬菌体核酸检测病原菌及其耐药性的新方法。以金黄色葡萄球菌、鲍曼不动杆菌与结核菌为研究对象，从各种样本中筛选三种病原菌广谱且高效的噬菌体；利用全基因组测序技术与生物信息学分析噬菌体序列，设计其特异性引物与探针；基于噬菌体宿主特异性与“指数”爆发的特点，通过定量PCR/微滴数字PCR检测噬菌体核酸拷贝数目变化从而实现病原菌的快速检测；基于噬菌体只在活菌中增殖的特点，利用建立的病原菌识别新方法进行药敏检测。本课题将建立一种新的病原菌及其耐药性快速检测方法，具有显著的社会效益和市场前景。

不断增加耐药性细菌的出现为目前病原菌感染治疗带来了巨大的挑战，病原菌及其耐药性的快速检测对于病人诊疗、控制耐药菌株的传播有着重要价值。传统的培养方法是病原菌鉴定与药敏检测的金标准，但是其培养时间长、易污染且工作量大。分子方法可以快速识别病原菌，但是其药敏检测局限于耐药机制已知位点的检测。噬菌体独特的宿主特异性、“指数”爆发以及仅仅在活菌中增殖的特点使得其在病原菌检测与药敏检测当中有着巨大的应用前景。在本研究，我们针对多重耐药鲍曼不动杆菌与耐甲氧西林金黄色葡萄球菌分离了多种噬菌体。利用全基因组测序技术我们完成了多重耐药鲍曼不动杆菌噬菌体p53、YC#06、pB3074以及耐甲氧西林金黄色葡萄球菌噬菌体pN315的测序；基于p53发展了噬菌体溶液当中噬菌体实时定量与复杂临床样本当中鲍曼不动杆菌快速检测的方法；完成了基于噬菌体核酸检测的多重PCR鉴定细菌与药敏的检测方法；基于多重耐药鲍曼不动杆菌噬菌体YC#06，进一步证明了噬菌体-抗生素联合使用对治疗耐多药鲍曼不动杆菌或其它耐药细菌感染潜在临床应用价值；我们的研究建立了一种新的病原菌及其耐药性快速检测方法，发现了噬菌体和抗生素联合使用对临床上十分棘手的多重耐药菌存在协同抗菌作用，具有重要的临床应用价值与显著的社会效益。