基于噬菌体识别机制的病原菌及其耐药性检测新方法

Bacterial detection and antimicrobial resistance test show great significance in diagnosis and treatment of infectious disease since they can improve the therapeutic efficacy and delay the onset of resistance resulting from abuse of broad-spectrum antibiotics. The reported protocols for these purposes often suffer from low efficiency and unsufficient specificity. In this project, virulent phages will be adopted to establish bioluminescent and chemiluminescent protocols with ideal specificity and high efficiency for bacterial detection and antimicrobial resistance test based on their recognition of host bacteria and reproduction mechanism. Firstly, bioluminescent protocol for bacterial detection will be developed based on the specific disruption of host pathogenic bacteria by phages. Secondly, phage tail fiber protein will be expressed and act as the recognition agent to establish sandwich protocol for chemiluminescent detection of bacteria. Thirdly, based on the fact that phages only reproduce in viable bacteria, antimicrobial resistance test protocol will be developed for bacteria in planktonic state. Lastly, the above developed antimicrobial resistance test protocol will also be extended to bacterial biofilm. This project can achieve facile and rapid bacterial identification, bacterial quantification and antimicrobial resistance test, thus provide efficient, sensitive and reliable assay platform for diagnosis and treatment of infectious diseases, especially for infectious diseases resulting from drug-resistant bacteria.

病原菌及其耐药性的检测对于感染性疾病的诊断和治疗具有重要意义，有助于提高此类疾病的治疗效果，并防止广谱抗生素滥用导致的细菌耐药问题。为解决已有方法在检测速度和特异性上的缺陷，本项目拟基于烈性噬菌体对于宿主菌的高特异性识别作用及其繁殖机制，研究病原菌及其耐药性的生物发光/化学发光检测方法。具体包括：①以烈性噬菌体作为病原菌识别物质，利用其繁殖过程对于病原菌的特异性裂解作用，建立病原菌生物发光检测方法。②表达噬菌体尾丝蛋白，将其作为病原菌分子识别试剂，基于双位点夹心模式建立病原菌化学发光检测方法。③利用噬菌体只在活菌中繁殖这一生物学特性，建立浮游状态病原菌耐药性检测方法。④模拟制备病原菌生物被膜，利用上述建立的耐药性检测方法，建立被膜状态病原菌耐药性检测方法。本研究有望简单快速地实现病原菌的鉴定、定量检测与耐药性评价，为感染性疾病尤其是耐药菌感染性疾病的诊疗提供高效、灵敏、可靠的分析方法平台。

广谱抗生素的滥用导致各种耐药菌株的数量不断增加，耐受几乎所有抗生素的“超级细菌”不断见诸报端。面对耐药病原菌所导致的一系列公共卫生事件，只有实现病原菌及其耐药谱进行快速检测，才能帮助医疗机构尽快选定针对该菌的窄谱抗生素，对危重感染病人实行及时有效的医学干预，并避免产生广谱抗生素滥用导致的耐药菌株。本项目针对金黄色葡萄球菌、铜绿假单胞菌、不动鲍曼杆菌、肺炎克雷伯菌、耻垢分枝杆菌等常见病原菌，分离获得了多株烈性噬菌体。利用烈性噬菌体及其功能蛋白对宿主菌的特异性识别作用，建立了一系列病原菌的化学发光、生物发光、荧光检测新方法。同时利用噬菌体只识别活菌这一生物学特性，建立了病原菌耐药性快速检测新方法。依此建立的基于噬菌体识别与发光检测的病原菌及其耐药性检测方法模型，其灵敏度、特异性、分析速度和操作简便性等关键参数已可满足复杂样品分析、低含量样品分析、现场分析和快速筛查的需要。此外，我们还研究了噬菌体内溶素蛋白对超级耐药病原菌的抗菌作用，证实了其对耐药菌感染的治疗作用，为后续抗菌药物的研究开发提供了初步基础。以上工作为临床诊断、食品安全、环境卫生、生物反恐等领域提供了有力的检测方法平台。在项目经费的资助下，在Anal. Chem.、Biosens. Bioelectron.、ACS Appl. Mater. Interfaces等刊物上发表SCI论文25篇；申请发明专利1项；以第一完成人身份获得2018年度“重庆市自然科学奖”二等奖。