Marras Salvatore A E, Chen Liang, Shashkina Elena, Davidson Rebecca M, Strong Michael, Daley Charles L, Kreiswirth Barry N
Public Health Research Institute Center, New Jersey Medical School, Rutgers University, Newark, New Jersey, USA.
Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, New Jersey, USA.
J Clin Microbiol. 2021 Jul 19;59(8):e0045521. doi: 10.1128/JCM.00455-21.
Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterial species that comprises three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. These predominantly environmental microorganisms have emerged as life-threatening chronic pulmonary pathogens in both immunocompetent and immunocompromised patients, and their acquisition of macrolide resistance due to the (41) gene and mutations in the 23S gene has dramatically impacted patient outcome. However, standard microbiology laboratories typically have limited diagnostic tools to distinguish M. abscessus subspecies, and the testing for macrolide resistance is often not done. Here, we describe the development of a real-time multiplex assay using molecular beacons to establish a robust, rapid, and highly accurate method to both distinguish M. abscessus subspecies and to determine which strains are susceptible to macrolides. We report a bioinformatic approach to identify robust subspecies sequence targets, the design and optimization of six molecular beacons to identify all genotypes, and the development and application of a 2-tube 3-color multiplex assay that can provide clinically significant treatment information in less than 3 h.
脓肿分枝杆菌是一种快速生长的非结核分枝杆菌，包括三个亚种：脓肿分枝杆菌脓肿亚种、脓肿分枝杆菌马赛亚种和脓肿分枝杆菌博列蒂亚种。这些主要存在于环境中的微生物已成为免疫功能正常和免疫功能低下患者中危及生命的慢性肺部病原体，并且它们因（41）基因和23S基因中的突变而获得大环内酯耐药性，这极大地影响了患者的治疗结果。然而，标准微生物实验室通常用于区分脓肿分枝杆菌亚种的诊断工具有限，而且大环内酯耐药性检测往往未进行。在此，我们描述了一种使用分子信标开发的实时多重检测方法，以建立一种强大、快速且高度准确的方法，既能区分脓肿分枝杆菌亚种，又能确定哪些菌株对大环内酯敏感。我们报告了一种生物信息学方法，用于识别强大的亚种序列靶点，设计和优化六种分子信标以识别所有基因型，以及开发和应用一种双管三色多重检测方法，该方法可在不到3小时内提供具有临床意义的治疗信息。
