Rapid, whole genome sequencing-based diagnosis of drug-resistant Mycobacterium abscessus complex and new options for treatment

非结核分枝杆菌（NTM）是结核分枝杆菌复合体和麻风分枝杆菌以外的分枝杆菌种类。它们在环境中无处不在，往往与水、土壤和医院病房隔绝。随着免疫功能低下患者（包括艾滋病毒感染者和血液病患者）以及囊性纤维化和慢性肺部疾病患者数量的增加，NTM作为人类疾病，特别是肺部疾病的病因的作用已经变得明显，最近的报告表明世界范围内的增加。特别是，脓肿分枝杆菌复合体包括一组快速生长的、耐多药的、非结核分枝杆菌，它们可引起广泛的皮肤和软组织疾病、中枢神经系统感染、菌血症、眼部和其他感染。目前的治疗指南基于有限的数据，这些数据主要来自专家意见、病例系列和少数随机临床试验。众所周知，脓肿分枝杆菌引起的感染很难治疗。虽然没有标准的治疗方法，但指南建议将大环内酯类药物与静脉注射的其他抗菌药物联合使用；然而，该方案已被证明对患者有很大的副作用。此外，药敏试验存在问题，在标准化方法上仍未达成共识，而且在实验室测量的药物敏感性与在患者身上观察到的药物活性之间存在重要差异。尽管最近的研究表明一些极好的活性，但没有在参比实验室常规检测抗结核新细菌药物（如贝达喹啉、delamanid）和用于耐药结核病病例的其他化合物（如氯法嗪）。全基因组测序（WGS）已广泛应用于临床场景，英国是世界上第一个在全国范围内率先将其用于结核分枝杆菌诊断、耐药性检测和分型的国家。这大大缩短了最终诊断的时间，一线易感性数据只需8天（从数周到有时数月）。然而，NTM的研究已经进行了很长时间，为了能够通过易感试验进行快速诊断，还需要做很多工作。亚种分化的进展使脓疡分枝杆菌复合体引起的肺部疾病得到更有效的治疗。例如，与脓肿分枝杆菌不同。脓肿，脓肿分枝。马氏杆菌对克拉霉素没有诱导抗性。导致大环内酯类药物耐药的erm基因的发现，是快速诊断如何使医生能够自信地使用克拉霉素并尽早优化治疗的一个例子。然而，脓肿分枝杆菌亚sp。脓肿仍然是诊断和治疗的挑战，因为80%的分离株对大环内酯耐药。目前尚无快速诊断的检测方法，对最佳抗菌药物和联合治疗缺乏共识，再加上可用药物匮乏。因此，需要进一步研究脓肿分枝杆菌的快速诊断和治疗方案。本研究项目的主要目的是研究WGS在脓肿分枝杆菌复合体中的应用，特别关注耐药性突变的快速检测（从而允许早期优化治疗）。第二个目的是研究各种化合物对脓疡分枝杆菌复合物的抗菌活性，包括抗微生物肽和噬菌体（从而为治疗耐药性提供额外的选择）。

Nontuberculous mycobacteria (NTM) are mycobacterial species other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. They are ubiquitous in the environment and often isolated from water, soil, and hospital wards. With increasing numbers of immunocompromised patients (including those with HIV infection and haematological disorders), as well as patients with cystic fibrosis and chronic lung disorders, the role of NTM as a cause of human, and in particular pulmonary, disease has become apparent, with recent reports indicating a worldwide increase. In particular, Mycobacterium abscessus complex comprises a group of rapidly growing, multidrug-resistant, nontuberculous mycobacteria that are responsible for a wide spectrum of skin and soft tissue diseases, central nervous system infections, bacteraemia, and ocular and other infections.Current treatment guidelines are based on limited data derived mostly from expert opinion, case series, and few randomized clinical trials. Infections caused by M. abscessus complex are notoriously difficult to treat. Although there is no standard treatment, the guidelines suggest the administration of macrolide-based therapy in combination with other antimicrobial agents administered intravenously; however, this regimen has been shown to have substantial side effects for patients. Additionally, susceptibility testing is problematic and there is still no consensus on a standardized method and there are important discrepancies between drug susceptibility measured in the laboratory and the activity of the drug observed in patients. New antimycobacterial drugs (i.e. bedaquiline, delamanid) and other compounds used in case of drug resistant tuberculosis (i.e. cloafazimine) are not routinely tested against NTM in the reference laboratory, despite recent research studies suggesting some excellent activity. Whole genome sequencing (WGS) has been applied to a wide range of clinical scenarios and England is the first country in the world to pioneer its use on a national scale for the diagnosis of M. tuberculosis, detection of drug resistance, and typing. This has drastically reduced the time to final diagnosis with first line susceptibilities data available in only 8 days (from weeks and sometime months). However, NTM have long been under investigated and much work still needs to be done to allow a rapid diagnosis with susceptibility testing. The advancement of subspecies differentiation has allowed for more effective management of pulmonary disease caused by M. abscessus complex. For example, unlike M. abscessus subsp. abscessus, M. abscessus subsp. massiliense does not have inducible resistance to clarithromycin. The discovery of the erm gene, which is responsible for macrolide resistance, is an example of how rapid diagnosis would enable the physician to confidently administer clarithromycin and optimize treatment early. However, M. abscessus subsp. abscessus still remains both a diagnostic and treatment challenge as 80% of isolates are macrolide resistant. There is no current test to allow a rapid diagnosis and there is a lack of consensus on the optimal antimicrobial agents and combination therapy, combined with the paucity of available drugs. Hence, further research in both rapid diagnosis and treatment options of M. abscessus is needed. The main aim of this research project is to investigate the utility of WGS applied to M. abscessus complex, with a particular focus on rapid detection of drug resistance mutations (thus, allowing early optimization of treatment). The secondary aim is to investigate the antimicrobial activity of various compounds against M. abscessus complex, including anti-microbial peptides and bacteriophages (thus, providing additional options to treat drug resistance).