In vitro synergy between manuka honey and amikacin against Mycobacterium abscessus complex shows potential for nebulisation therapy.

Mycobacterium abscessusis an opportunistic human pathogen of increasing concern, due to its ability to cause aggressive pulmonary infections (especially in cystic fibrosis patients), as well as skin and soft tissue infections. M. abscessus is intrinsically drug resistant and treatment regimens are lengthy, consisting of multiple antibiotics with severe side effects and poor patient success rates. New and novel strategies are urgently required to combat these infections. One such strategy thus far overlooked for mycobacteria is manuka honey. For millennia manuka honey has been shown to have wide ranging medicinal properties, which have more recently been identified for its broad spectrum of antimicrobial activity. Here we demonstrate that manuka honey can be used to inhibit M. abscessus and a variety of drug resistant clinical isolates in vitro. We also demonstrate using a microbroth dilution checkerboard assay that manuka honey works synergistically with amikacin, which is one of the current front line antibiotics used for treatment of M. abscessus infections. This was further validated using an in vitro inhalation model, where we showed that with the addition of manuka honey, the amikacin dosage can be lowered whilst increasing its efficacy. These findings demonstrate the utility of manuka honey for incorporation into nebulised antibiotic treatment for respiratory infections, in particular M. abscessus. These results pave the way for a change of strategy for M. abscessus management, offering new therapeutic options for this deadly infection.

Cholesterol-dependent activity of dapsone against non-replicating persistent mycobacteria.

One-third of the world's population is estimated to be latently infected with Mycobacterium tuberculosis. This reservoir of bacteria is largely resistant to antimicrobial treatment that often only targets actively replicating mycobacteria, with current treatment for latent infection revolving around inhibiting the resuscitation event rather than preventing or treating latent infection. As a result, antimicrobials that target latent infection often have little to no activity in vivo. Here we report a method of in vitro analysis of physiologically relevant non-replicating persistence (NRP) utilizing cholesterol as the sole carbon source, alongside hypoxia as a driver of Mycobacterium bovis BCG into the NRP state. Using the minimal cholesterol media NRP assay, we observed an increased state of in vitro resistance to front-line anti-tubercular compounds. However, following a phenotypic screen of an approved-drug library, we identified dapsone as a bactericidal active molecule against cholesterol-dependent NRP M. bovis BCG. Through an overexpression trial of probable antimicrobial target enzymes, we further identified FolP2, a non-functional dihydropteroate synthase homologue, as the likely target of dapsone under cholesterol-NRP due to a significant increase in bacterial resistance when overexpressed. These results highlight the possible reason for little in vivo activity seen for current front-line anti-NRP drugs, and we introduce a new methodology for future drug screening as well as a potential role for dapsone inclusion within the current treatment regime.

Assembly of the Mycobacterial Cell Wall.

Mycobacterium tuberculosis remains one of the most successful bacterial pathogens, claiming over 1.3 million lives worldwide in 2013. The emergence of multidrug-resistant and extensively drug-resistant isolates has prompted the need for new drugs and drug targets. M. tuberculosis possesses an unusual cell wall dominated by lipids and carbohydrates that provides a permeability barrier against hydrophilic drugs and is crucial for its survival and virulence. This large macromolecular structure, termed the mycolyl-arabinogalactan-peptidoglycan complex, and the phosphatidyl-myo-inositol-based lipoglycans are key features of the mycobacterial cell wall. Assembly of these cell wall components is an attractive target for the development of chemotherapeutics against tuberculosis. Herein, we focus on recent biochemical and molecular insights into these complex molecules of M. tuberculosis cell wall.

Manuka honey in combination with azithromycin shows potential for improved activity against Mycobacterium abscessus.

Mycobacterium abscessus is an increasingly prevalent opportunistic pathogen causing both pulmonary and skin and soft tissue infections. It is of increasing concern for immunocompromised individuals, such as those with cystic fibrosis, due to its highly drug resistant nature and ability to evade the host immune system. Current treatments for M. abscessus pulmonary infections are largely ineffective and treatment outcomes are generally poor, thus we urgently require new treatments to combat these infections. Recently, it has been demonstrated that manuka honey is effective against M. abscessus and can improve the inhibitory effect of amikacin. Here, we explore the potential improvement of both azithromycin and tobramycin with the addition of manuka honey against M. abscessus complex. Improved growth inhibition was observed for azithromycin with manuka honey against all M. abscessus subspecies. Improved bactericidal activity was also observed. Importantly, the macrolide resistant M. abscessus subsp. bolletii showed improved inhibition and bactericidal activity was obtained in response to 0.117 g/mL manuka honey MGO40 with 16 µg/mL azithromycin. No improved activity was observed for tobramycin and manuka honey against any of the M. abscessus isolates tested. This demonstrates the potential for antibiotic enhancement by the addition of manuka honey, furthering the applications of therapeutic manuka honey.

Indole-containing arene-ruthenium complexes with broad spectrum activity against antibiotic-resistant bacteria.

Antimicrobial resistant (AMR) bacteria are emerging and spreading globally, threatening our ability to treat common infectious diseases. The development of new classes of antibiotics able to kill or inhibit the growth of such AMR bacteria through novel mechanisms of action is therefore urgently needed. Here, a new family of indole-containing arene ruthenium organometallic compounds are screened against several bacterial species and drug resistant strains. The most active complex [(p-cym)Ru(O-cyclohexyl-1H-indole-2-carbothioate)Cl] (3) shows growth inhibition and bactericidal activity against different organisms (Acinetobacter baumannii, Mycobacterium abscessus, Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica serovar Typhi and Escherichia coli), demonstrating broad-spectrum inhibitory activity. Importantly, this compound series exhibits low toxicity against human cells. Owing to the novelty of the antibiotic family, their moderate cytotoxicity, and their inhibitory activity against Gram positive, Gram negative and acid-fast, antibiotic resistant microorganisms, this series shows significant promise for further development.

Drug Susceptibility Screening Using In Vitro Models of Hypoxic Non-Replicating Persistent Mycobacteria.

Non-replicating persistence (NRP) is a functional adaptation that mycobacteria undergo in response to the stresses of the granuloma, facilitating antibiotic tolerance and long-term infection. These stresses, or NRP-inducing factors, include hypoxia, nutrient deprivation, and nitric oxide assault, which mycobacteria are well evolved to tolerate through a series of metabolic and physiological adaptations producing the NRP state. Most attempts to replicate these conditions in vitro have focused on only one of these factors at a time for ease and simplicity, but as a result, do not necessarily produce physiologically relevant phenotypes. Here, we provide the methods for two different in vitro NRP strategies that are useful for drug susceptibility testing and high-throughput screening.

In vitro efficacy of relebactam versus avibactam against Mycobacterium abscessus complex.

Infections resulting from Mycobacterium abscessus are increasing in prevalence worldwide, with the greatest risk posed to patients with underlying respiratory conditions. Treatment for infections is difficult due to wide ranging intrinsic antimicrobial resistance, which is compounded by the existence of a range of subspecies within the M. abscessus complex, each with varying additional antimicrobial resistance profiles. Previously, the use of ß-lactam/ß-lactamase inhibitors within a combination therapy has been proposed as an effective treatment option for pulmonary M. abscessus infections. Here, we assess the in vitro efficacy of two non-ß-lactam based inhibitors, relebactam and avibactam, as agents against M. abscessus with their respective partner drugs imipenem and ceftazidime, as well as in triplicate combinations with additional ß-lactam antibiotics against the M. abscessus complex. We have shown that the commercially available ratio of imipenem to relebactam is the appropriate ratio for bactericidal activity against M. abscessus, whereas the ratio between ceftazidime and avibactam is redundant, due to inactivity of ceftazidime to inhibit the bacteria. We have identified that the use of imipenem and meropenem alongside either relebactam or avibactam yield low minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for each M. abscessus subspecies, which are within the therapeutically achievable concentration ranges within the epithelial lining fluid of the lungs. We propose the implementation of imipenem with relebactam in place of stand-alone imipenem into the current treatment regime, alongside meropenem, as a future front-line treatment option for M. abscessus complex infections.

Microfluidics as a Novel Technique for Tuberculosis: From Diagnostics to Drug Discovery.

Tuberculosis (TB) remains a global healthcare crisis, with an estimated 5.8 million new cases and 1.5 million deaths in 2020. TB is caused by infection with the major human pathogen Mycobacterium tuberculosis, which is difficult to rapidly diagnose and treat. There is an urgent need for new methods of diagnosis, sufficient in vitro models that capably mimic all physiological conditions of the infection, and high-throughput drug screening platforms. Microfluidic-based techniques provide single-cell analysis which reduces experimental time and the cost of reagents, and have been extremely useful for gaining insight into monitoring microorganisms. This review outlines the field of microfluidics and discusses the use of this novel technique so far in M. tuberculosis diagnostics, research methods, and drug discovery platforms. The practices of microfluidics have promising future applications for diagnosing and treating TB.

Clinical Significance of Manuka and Medical-Grade Honey for Antibiotic-Resistant Infections: A Systematic Review.

Antimicrobial resistance is an ever-increasing global issue that has the potential to overtake cancer as the leading cause of death worldwide by 2050. With the passing of the "golden age" of antibiotic discovery, identifying alternative treatments to commonly used antimicrobials is more important than ever. Honey has been used as a topical wound treatment for millennia and more recently has been formulated into a series of medical-grade honeys for use primarily for wound and burn treatment. In this systematic review, we examined the effectiveness of differing honeys as an antimicrobial treatment against a variety of multidrug-resistant (MDR) bacterial species. We analysed 16 original research articles that included a total of 18 different types of honey against 32 different bacterial species, including numerous MDR strains. We identified that Surgihoney was the most effective honey, displaying minimum inhibitory concentrations as low as 0.1% (w/v); however, all honeys reviewed showed a high efficacy against most bacterial species analysed. Importantly, the MDR status of each bacterial strain had no impact on the susceptibility of the organism to honey. Hence, the use of honey as an antimicrobial therapy should be considered as an alternative approach for the treatment of antibiotic-resistant infections.

Author Correction: Effect of Amoxicillin in combination with Imipenem-Relebactam against Mycobacterium abscessus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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 ß-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 ß-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.

Development of a novel secondary phenotypic screen to identify hits within the mycobacterial protein synthesis pipeline.

BACKGROUND: Whole-cell phenotypic screening is the driving force behind modern anti-tubercular drug discovery efforts. Focus has shifted from screening for bactericidal scaffolds to screens incorporating target deconvolution. Target-based screening aims to direct drug discovery toward known effective targets and avoid investing resources into unproductive lines of enquiry. The protein synthesis pipeline, including RNA polymerase and the ribosome, is a clinically proven target in Mycobacterium tuberculosis. Screening for new hits of this effective target pathway is an invaluable tool in the drug discovery arsenal. METHODS: Using M. tuberculosis H37Rv augmented with anhydrotetracycline-inducible expression of mCherry, a phenotypic screen was developed for the identification of protein synthesis inhibitors in a medium throughput screening format. RESULTS: The assay was validated using known inhibitors of protein synthesis to show a dose-dependent reduction in mCherry fluorescence. This was expanded to a proprietary screen of hypothetical protein synthesis hits and modified to include quantitative viability measurement of cells using resazurin. CONCLUSION: Following the success of the proprietary screen, a larger scale screen of the GlaxoSmithKline anti-tubercular library containing 2799 compounds was conducted. Combined single shot and dose-response screening yielded 18 hits, 0.64% of all screened compounds.

Aston University's Antimicrobial Resistance (AMR) Roadshow: raising awareness and embedding knowledge of AMR in key stage 4 learners.

Antimicrobial resistance (AMR) is a global healthcare problem and therefore raising awareness within young learners is imperative. An AMR roadshow was designed to take key stage 4 students' learning 'out of the classroom', assess pre-existing knowledge of AMR and determine the impact of the roadshow on knowledge retention. Knowledge and subsequent retention were measured pre- and post-event through a standardised questionnaire. The roadshow significantly improved knowledge and understanding of AMR, which was retained for a minimum of twelve weeks. Engaging and interactive strategies addressing key health issues provide a positive learning experience which contributes to retained knowledge in young learners.

Changing the Rules of TB-Drug Discovery.

The discovery of new drugs with novel targets is paramount to the continued success of tuberculosis (TB) treatment due to the increasing prevalence of antibiotic resistant infections in the TB population. Mycobacterium tuberculosis (Mtb) fumarate hydratase (fumarase) is a highly conserved essential protein that shares an active site with human fumarase, making active site inhibition equally cytotoxic for both bacteria and humans. The recent discovery of a set of new Mtb inhibitory compounds that target Mtb-fumarase by binding to a nonconserved allosteric site is a major advancement, providing further evidence to dispel the antibiotic discovery dogma that conserved proteins do not make good antibiotic targets.

Dissecting the Antimicrobial Composition of Honey.

Honey is a complex sweet food stuff with well-established antimicrobial and antioxidant properties. It has been used for millennia in a variety of applications, but the most noteworthy include the treatment of surface wounds, burns and inflammation. A variety of substances in honey have been suggested as the key component to its antimicrobial potential; polyphenolic compounds, hydrogen peroxide, methylglyoxal and bee-defensin 1. These components vary greatly across honey samples due to botanical origin, geographical location and secretions from the bee. The use of medical grade honey in the treatment of surface wounds and burns has been seen to improve the healing process, reduce healing time, reduce scarring and prevent microbial contamination. Therefore, if medical grade honeys were to be included in clinical treatment, it would reduce the demand for antibiotic usage. In this review, we outline the constituents of honey and how they affect antibiotic potential in a clinical setting. By identifying the key components, we facilitate the development of an optimally antimicrobial honey by either synthetic or semisynthetic production methods.

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.

Crystal structure of Mycobacterium tuberculosis FadB2 implicated in mycobacterial ß-oxidation.

The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in ß-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosis L-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Šresolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD+-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA-FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.

Modelling a Silent Epidemic: A Review of the In Vitro Models of Latent Tuberculosis.

Tuberculosis (TB) is the primary cause of death by a single infectious agent; responsible for around two million deaths in 2016. A major virulence factor of TB is the ability to enter a latent or Non-Replicating Persistent (NRP) state which is presumed untreatable. Approximately 1.7 billion people are latently infected with TB and on reactivation many of these infections are drug resistant. As the current treatment is ineffective and diagnosis remains poor, millions of people have the potential to reactivate into active TB disease. The immune system seeks to control the TB infection by containing the bacteria in a granuloma, where it is exposed to stressful anaerobic and nutrient deprived conditions. It is thought to be these environmental conditions that trigger the NRP state. A number of in vitro models have been developed that mimic conditions within the granuloma to a lesser or greater extent. These different models have all been utilised for the research of different characteristics of NRP Mycobacterium tuberculosis, however their disparity in approach and physiological relevance often results in inconsistencies and a lack of consensus between studies. This review provides a summation of the different NRP models and a critical analysis of their respective advantages and disadvantages relating to their physiological relevance.

Antimycobacterial drug discovery using Mycobacteria-infected amoebae identifies anti-infectives and new molecular targets.

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK.

The 'Antibiotic Apocalypse' - Scaremongering or Scientific Reporting?

Antimicrobial resistance is dominating scientific media. We are warned of an impending 'antibiotic apocalypse', where mankind faces its biggest threat, untreatable microbes. However, the world is not ending. Scientists are responding to the threat; new knowledge and chemotherapeutics are being created to safeguard our future. The future is bright, not gloomy.