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

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).

The project will include different phases:
1. Selection of resistant laboratory mutants (objective 1) - M. abscessus resistant strains to a single drug will be selected in the laboratory and WGS performed on the resistant isolates. This will allow determining the genetic mutations conferring resistance by applying a process previously described in other mycobacteria, both in M. smegmatis and M. tuberculosis.
2. Next generation sequencing for rapid diagnosis of resistance (objective 2)- Once a set of mutations has been identified in the laboratory mutants, whole genome sequencing will also be performed on resistant clinical isolates to validate the susceptibility profile with the genetic mutations for rapid diagnosis. Susceptibility data to first line antibiotics for all our M. abscessus complex isolates are already available as it was performed as part of the routine clinical service.
3. Susceptibility testing of clinical isolates (objective 3) - Our research collaborators at the National Heart and Lung Institute have an extensive collection of NTM clinical strains (over a thousand) and selected M. abscessus complex isolates from this collection will be tested against various compounds (and combination of drugs) to assess their susceptibility profile to new antibiotics (for example, bedaquiline and delamanid), currently not included in any first line testing. Testing will be performed using a combination of different methods (resazurin and other dilution techniques) to assess the validity and reproducibility of such methods. Additionally, our collaborators from the MRC Centre for Molecular Bacteriology and Infection are already conducting research on new anti-microbial peptides with promising antimycobacterial effect but such compounds have yet to be tested against M. abscessus resistant strains. To complete the picture, bacteriophages from our research partners at the University of Pittsburgh will be tested against such resistant strains.

This proposal addresses the challenges of M. abscessus infections through a multifaced approach, including rapid diagnosis and susceptibility testing of new compounds. The potential outcomes for the research are far reaching, and will influence a wide range of researchers, clinicians, policy makers, industry and the public. The main impact from this research will be in the following areas:
- Societal benefit: drug resistance in M. asbscessus is becoming particularly problematic and there are very limited treatment options. Some benefits may be relatively immediate for patients as the combination treatment with some of the drugs tested (i.e. bedaquiline and delamanid) or even bacteriophages can be easily implemented in clinical practice.
- Translational research: Imperial College London is currently a world leader in antimicrobial resistance; this project will build upon and be supported by extensive interdisciplinary collaborations. A wide range of stakeholders will benefit from this research including academic researchers (both clinical and non-clinical), healthcare providers, policy makers, industry and patients (as mentioned above). All the data gathered will be useful for the creation of a database that will constitute the base of future computer software for the rapid determination of resistance in M. abscessus and other NTMs. Other researchers around the world and diagnostic companies will be able to access such database and the project results.
- Scientific and academic dissemination: the project is expected to result in high impact publications and the results will also be presented at national and international conferences.
- Communications, engagement and society: Professor Holmes' research team and our collaborators recognise the importance of patient involvement in directing the development and adoption of research. They have extensive experience in Public Patient Involvement (PPI) activities. The project will be supported by the HPRU PPI committee and a patient representative. The team has also performed and published extensive work on patient engagement.
Finally, the project is expected to improve the cross-disciplinary collaborations between academic researchers and clinicians by creating an innovative new partnership, linking together world renown research centres and NHS partners, opening new opportunities of future research projects.