Investigating Mycobacterium abscessus to inform treatment and drug discovery

The environmental non-tuberculous mycobacteria, Mycobacterium abscessus, is quickly becoming a major health concern in developed countries in part to its extensive multi-drug resistance. Those at risk of contracting M. abscessus lung disease are primarily people who are immunocompromised or have pre-existing lung disorders such as Cystic Fibrosis. Treatment for this disease involves a lengthy regimen of several antibiotics, despite this, treatment failure rates remain unacceptably high. The background of this infectious disease, its clinical manifestation and management is discussed in detail in Chapter 1. The first results chapter of this thesis, chapter 2, makes use of genomic techniques to explore the subspecies of a panel of M. abscessus clinical isolates, alongside their drug susceptibility patterns, in order to elucidate a link between M. abscessus subspecies (M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense) and drug resistance. Given the high levels of treatment failure, it is essential to find new treatments for M. abscessus infections, that can be introduced into clinical practice in the near future. Chapter 3 of this thesis explores the repurposing of a new and approved β-lactamase inhibitor, relebactam, for inclusion into the M. abscessus chemotherapeutic regimen. Here, relebactam’s efficacy against the endogenous M. abscessus β-lactamase, BlaMab is discovered, as well as its inhibitory activity when combined with the carbapenem, imipenem. This activity is further potentiated with by the addition of amoxicillin. This three-drug combination has widespread activity against a panel of clinical isolates, within a therapeutically achievable concentration range. Finally, an in vitro model of persistence in M. abscessus infection was developed and this was used to assess frontline drug susceptibilities, providing an insight into the possible causes of treatment failure for this infection. Overall, this body of work contributes to the knowledge of the organism, provides a greater understanding of the clinical challenge it represents and proposes a new treatment option for patients suffering with this deadly infection

Mycobacterium abscessus: Environmental Bacterium Turned Clinical Nightmare

Mycobacteria are a large family of over 100 species, most of which do not cause diseases in humans. The majority of the mycobacterial species are referred to as nontuberculous mycobacteria (NTM), meaning they are not the causative agent of tuberculous (TB) or leprosy, i.e., Mycobacterium tuberculous complex and Mycobacterium leprae, respectively. The latter group is undoubtedly the most infamous, with TB infecting an estimated 10 million people and causing over 1.2 million deaths in 2017 alone TB and leprosy also differ from NTM in that they are only transmitted from person to person and have no environmental reservoir, whereas NTM infections are commonly acquired from the environment. It took until the 1950′s for NTM to be recognised as a potential lung pathogen in people with underlying pulmonary disease and another three decades for NTM to be widely regarded by the medical community when Mycobacterium avium complex was identified as the most common group of opportunistic pathogens in AIDS patients. This review focuses on an emerging NTM called Mycobacterium abscessus (M. abs). M. abs is a rapidly growing NTM that is responsible for opportunistic pulmonary infections in patients with structural lung disorders such as cystic fibrosis and bronchiectasis, as well as a wide range of skin and soft tissue infections in humans. In this review, we discuss how we came to understand the pathogen, how it is currently treated and examine drug resistance mechanisms and novel treatments currently in development. We highlight the urgent need for new and effective treatments for M. abs infection as well as improved in vivo methods of efficacy testing

Effect of Amoxicillin in combination with Imipenem-Relebactam against Mycobacterium abscessus

Infections caused by Mycobacterium abscessus are increasing in prevalence in cystic fibrosis patients. This opportunistic pathogen′s intrinsic resistance to most antibiotics has perpetuated an urgent demand for new, more effective therapeutic interventions. Here we report a prospective advance in the treatment of M. abscessus infection; increasing the susceptibility of the organism to amoxicillin, by repurposing the β-lactamase inhibitor, relebactam, in combination with the front line M. abscessus drug imipenem. We establish by multiple in vitro methods that this combination works synergistically to inhibit M. abscessus. We also show the direct competitive inhibition of the M. abscessus β-lactamase, BlaMab, using a novel assay, which is validated kinetically using the nitrocefin reporter assay and in silico binding studies. Furthermore, we reverse the susceptibility by overexpressing BlaMab in M. abscessus, demonstrating relebactam-BlaMab target engagement. Finally, we highlight the in vitro efficacy of this combination against a panel of M. abscessus clinical isolates, revealing the therapeutic potential of the amoxicillin-imipenem-relebactam combination

Remdesivir-ivermectin combination displays synergistic interaction with improved in vitro activity against SARS-CoV-2

A key element for the prevention and management of COVID-19 is the development of effective therapeutics. Drug combination strategies of repurposed drugs offer several advantages over monotherapies, including the potential to achieve greater efficacy, the potential to increase the therapeutic index of drugs and the potential to reduce the emergence of drug resistance. Here, we report on the in vitro synergistic interaction between two FDA approved drugs, remdesivir and ivermectin resulting in enhanced antiviral activity against SARS-CoV-2. Whilst the in vitro synergistic activity reported here does not support the clinical application of this combination treatment strategy, due to insufficient exposure of ivermectin in vivo, the data do warrant further investigation. Efforts to define the mechanisms underpinning the observed synergistic action, could lead to the development of novel therapeutic treatment strategies

Lack of antiviral activity of probenecid in vitro and in Syrian golden hamsters

Objectives Antiviral interventions are required to complement vaccination programmes and reduce the global burden of COVID-19. Prior to initiation of large-scale clinical trials, robust preclinical data to support candidate plausibility are required. This work sought to further investigate the putative antiviral activity of probenecid against SARS-CoV-2. Methods Vero E6 cells were preincubated with probenecid, or control media for 2 h before infection (SARS-CoV-2/Human/Liverpool/REMRQ0001/2020). Probenecid or control media was reapplied, plates reincubated and cytopathic activity quantified by spectrophotometry after 48 h. In vitro human airway epithelial cell (HAEC) assays were performed for probenecid against SARS-CoV-2-VoC-B.1.1.7 (hCoV-19/Belgium/rega-12211513/2020; EPI_ISL_791333, 2020-12-21) using an optimized cell model for antiviral testing. Syrian golden hamsters were intranasally inoculated (SARS-CoV-2 Delta B.1.617.2) 24 h prior to treatment with probenecid or vehicle for four twice-daily doses. Results No observable antiviral activity for probenecid was evident in Vero E6 or HAEC assays. No reduction in total or subgenomic RNA was observed in terminal lung samples (P > 0.05) from hamsters. Body weight of uninfected hamsters remained stable whereas both probenecid- and vehicle-treated infected hamsters lost body weight (P > 0.5). Conclusions These data do not support probenecid as a SARS-CoV-2 antiviral drug