Profiling the mycobacteria biofilm: a multidisciplinary mix of mutants and mass spectrometry

非结核分枝杆菌(NTM)，如脓肿分枝杆菌和嵌合体，可在免疫功能低下或患有慢性肺部疾病(如囊性纤维化)的患者中导致高死亡率和发病率。从环境蓄水池吸入后，这类患者的肺泡壁中会形成分枝杆菌生物膜。最近，从淋浴喷头和医院空调机组中分离出致病菌株。众所周知，生物膜很难根除，而且与抗菌素耐药性(AMR)的增加有关。脓肿和嵌合体感染的治疗通常需要2-3年的抗生素。我们想要解决的问题是，在NTM生物被膜形成过程中，AMR和治疗时间的增加是由于缺乏抗生素的渗透，导致非治疗性和AMR产生水平，还是由于表型和/或遗传耐药性的发展。在这个项目中，我们将使用Nano-SIMS(纳米级二次离子质谱仪)来测量用于治疗NTM感染的抗生素Bedaquiline(BDQ)对NTM生物膜(脓肿分支杆菌和嵌合体分支杆菌)单个细胞的渗透性。Nano-SIMS将不同离子的相对丰度映射到纳米级，并可用于通过生物膜测量剖面。此外，还将测量BDK的最低抑菌浓度(MIC)和最小杀灭时间(MDK)，以确定生物膜和浮游生长控制中的抗生素敏感性。了解在NTM生物膜中AMR的产生和长期治疗可能会导致死亡率和发病率的提高。开发新的治疗策略可以提高治疗效果，缩短治疗时间，促进AMR的发展。

Non-tuberculous mycobacteria (NTMs), such as Mycobacterium abscessus and chimaera, can cause high mortality and morbidity amongst patients who are immunocompromised or have chronic lung diseases, such as cystic fibrosis. Mycobacterial biofilms can form in the alveolar walls of such patients following inhalation from environmental reservoirs. Recently, pathogenic strains have been isolated from shower heads and hospital air conditioning units. Biofilms are notoriously difficult to eradicate and are associated with the development of increased antimicrobial resistance (AMR). Treatment for M. abscessus and chimaera infections often requires 2-3 antibiotics over 2 years.The question we want to address is whether the increased AMR and treatment time in NTM biofilm formation is due to lack of antibiotic penetration, resulting in non-therapeutic and AMR-generative levels, or the development of phenotypic and/or genetic resistance. In this project we will use Nano-SIMS (nano-scale secondary ion mass spectrometry) to measure penetration of the antibiotic bedaquiline (BDQ), used to treat NTM infections, into individual cells of NTM biofilms (M.abscessus and M.chimaera). Nano-SIMS maps the relative abundance of different ions down to the nano-scale and can be used to measure in profile through the biofilm. In addition, the minimum inhibitory concentration (MIC) and minimum duration for killing (MDK) of BDK will be measured to determine antibiotic susceptibility in biofilms and a planktonic growth control. Understanding the AMR generation and prolonged treatment in NTM biofilms could lead to improved mortality and morbidity. The development of novel treatment strategies could enhance treatment efficacy, reduce treatment duration and AMR development.