Evaluation of critical parameters in the hollow-fibre system for tuberculosis: A case study of moxifloxacin.

AIMS: The hollow­fibre system for tuberculosis (HFS­TB) is a preclinical model qualified by the European Medicines Agency to underpin the anti­TB drug development process. It can mimic in vivo pharmacokinetic (PK)­pharmacodynamic (PD) attributes of selected antimicrobials, which could feed into in silico models to inform the design of clinical trials. However, historical data and published protocols are insufficient and omit key information to allow experiments to be reproducible. Therefore, in this work, we aim to optimize and standardize various HFS­TB operational procedures. METHODS: First, we characterized bacterial growth dynamics with different types of hollow­fibre cartridges, Mycobacterium tuberculosis strains and media. Second, we mimicked a moxifloxacin PK profile within hollow­fibre cartridges, in order to check drug­fibres compatibility. Lastly, we mimicked the moxifloxacin total plasma PK profile in human after once daily oral dose of 400 mg to assess PK­PD after different sampling methods, strains, cartridge size and bacterial adaptation periods before drug infusion into the system. RESULTS: We found that final bacterial load inside the HFS­TB was contingent on the studied variables. Besides, we demonstrated that drug­fibres compatibility tests are critical preliminary HFS­TB assays, which need to be properly reported. Lastly, we uncovered that the sampling method and bacterial adaptation period before drug infusion significantly impact actual experimental conclusions. CONCLUSION: Our data contribute to the necessary standardization of HFS­TB experiments, draw attention to multiple aspects of this preclinical model that should be considered when reporting novel results and warn about critical parameters in the HFS­TB currently overlooked.

Amoxicillin/clavulanate in combination with rifampicin/clarithromycin is bactericidal against Mycobacterium ulcerans.

BACKGROUND: Buruli ulcer (BU) is a skin neglected tropical disease (NTD) caused by Mycobacterium ulcerans. WHO-recommended treatment requires 8-weeks of daily rifampicin (RIF) and clarithromycin (CLA) with wound care. Treatment compliance may be challenging due to socioeconomic determinants. Previous minimum Inhibitory Concentration and checkerboard assays showed that amoxicillin/clavulanate (AMX/CLV) combined with RIF+CLA were synergistic against M. ulcerans. However, in vitro time kill assays (TKA) are a better approach to understand the antimicrobial activity of a drug over time. Colony forming units (CFU) enumeration is the in vitro reference method to measure bacterial load, although this is a time-consuming method due to the slow growth of M. ulcerans. The aim of this study was to assess the in vitro activity of RIF, CLA and AMX/CLV combinations against M. ulcerans clinical isolates by TKA, while comparing four methodologies: CFU enumeration, luminescence by relative light unit (RLU) and optical density (at 600 nm) measurements, and 16S rRNA/IS2404 genes quantification. METHODOLOGY/PRINCIPAL FINDINGS: TKA of RIF, CLA and AMX/CLV alone and in combination were performed against different M. ulcerans clinical isolates. Bacterial loads were quantified with different methodologies after 1, 3, 7, 10, 14, 21 and 28 days of treatment. RIF+AMX/CLV and the triple RIF+CLA+AMX/CLV combinations were bactericidal and more effective in vitro than the currently used RIF+CLA combination to treat BU. All methodologies except IS2404 quantitative PCR provided similar results with a good correlation with CFU enumeration. Measuring luminescence (RLU) was the most cost-effective methodology to quantify M. ulcerans bacterial loads in in vitro TKA. CONCLUSIONS/SIGNIFICANCE: Our study suggests that alternative and faster TKA methodologies can be used in BU research instead of the cumbersome CFU quantification method. These results provide an in vitro microbiological support to of the BLMs4BU clinical trial (NCT05169554, PACTR202209521256638) to shorten BU treatment.

In vitro synergy screens of FDA-approved drugs reveal novel zidovudine- and azithromycin-based combinations with last-line antibiotics against Klebsiella pneumoniae.

Treatment of infections caused by multi-drug resistant (MDR) enterobacteria remains challenging due to the limited therapeutic options available. Drug repurposing could accelerate the development of new urgently needed successful interventions. This work aimed to identify and characterise novel drug combinations against Klebsiella pneumoniae based on the concepts of synergy and drug repurposing. We first performed a semi-qualitative high-throughput synergy screen (sHTSS) with tigecycline, colistin and fosfomycin (last-line antibiotics against MDR Enterobacteriaceae) against a FDA-library containing 1430 clinically approved drugs; a total of 109 compounds potentiated any of the last-line antibiotics. Selected hits were further validated by secondary checkerboard (CBA) and time-kill (TKA) assays, obtaining 15.09% and 65.85% confirmation rates, respectively. Accordingly, TKA were used for synergy classification based on determination of bactericidal activities at 8, 24 and 48 h, selecting 27 combinations against K. pneumoniae. Among them, zidovudine or azithromycin combinations with last-line antibiotics were further evaluated by TKA against a panel of 12 MDR/XDR K. pneumoniae strains, and their activities confronted with those clinical combinations currently used for MDR enterobacteria treatment; these combinations showed better bactericidal activities than usual treatments without added cytotoxicity. Our studies show that sHTSS paired to TKA are powerful tools for the identification and characterisation of novel synergistic drug combinations against K. pneumoniae. Further pre-clinical studies might support the translational potential of zidovudine- and azithromycin-based combinations for the treatment of these infections.

Implementing best practices on data generation and reporting of Mycobacterium tuberculosis in vitro assays within the ERA4TB consortium.

Tuberculosis (TB) is the historical leading cause of death by a single infectious agent. The European Regimen Accelerator for Tuberculosis (ERA4TB) is a public-private partnership of 30+ institutions with the objective to progress new anti-TB regimens into the clinic. Thus, robust and replicable results across independent laboratories are essential for reliable interpretation of treatment efficacy. A standardization workgroup unified in vitro protocols and data reporting templates. Time-kill assays provide essential input data for pharmacometric model-informed translation of single agents and regimens activity from in vitro to in vivo and the clinic. Five conditions were assessed by time-kill assays in six independent laboratories using four bacterial plating methods. Baseline bacterial burden varied between laboratories but variability was limited in net drug effect, confirming 2.5 µL equally robust as 100 µL plating. This exercise establishes the foundations of collaborative data generation, reporting, and integration within the overarching Antimicrobial Resistance Accelerator program.

Repurposing ß-Lactams for the Treatment of Mycobacterium kansasii Infections: An In Vitro Study.

Mycobacterium kansasii (Mkn) causes tuberculosis-like lung infection in both immunocompetent and immunocompromised patients. Current standard therapy against Mkn infection is lengthy and difficult to adhere to. Although ß-lactams are the most important class of antibiotics, representing 65% of the global antibiotic market, they have been traditionally dismissed for the treatment of mycobacterial infections, as they were considered inactive against mycobacteria. A renewed interest in ß-lactams as antimycobacterial agents has shown their activity against several mycobacterial species, including M. tuberculosis, M. ulcerans or M. abscessus; however, information against Mkn is lacking. In this study, we determined the in vitro activity of several ß-lactams against Mkn. A selection of 32 agents including all ß-lactam chemical classes (penicillins, cephalosporins, carbapenems and monobactams) with three ß-lactamase inhibitors (clavulanate, tazobactam and avibactam) were evaluated against 22 Mkn strains by MIC assays. Penicillins plus clavulanate and first- and third-generation cephalosporins were the most active ß-lactams against Mkn. Combinatorial time-kill assays revealed favorable interactions of amoxicillin-clavulanate and cefadroxil with first-line Mkn treatment. Amoxicillin-clavulanate and cefadroxil are oral medications that are readily available, and well tolerated with an excellent safety and pharmacokinetic profile that could constitute a promising alternative option for Mkn therapy.

Comparison of 8 weeks standard treatment (rifampicin plus clarithromycin) vs. 4 weeks standard plus amoxicillin/clavulanate treatment [RC8 vs. RCA4] to shorten Buruli ulcer disease therapy (the BLMs4BU trial): study protocol for a randomized controlled multi-centre trial in Benin.

BACKGROUND: Buruli ulcer (BU) is a neglected tropical disease caused by Mycobacterium ulcerans that affects skin, soft tissues, and bones, causing long-term morbidity, stigma, and disability. The recommended treatment for BU requires 8 weeks of daily rifampicin and clarithromycin together with wound care, physiotherapy, and sometimes tissue grafting and surgery. Recovery can take up to 1 year, and it may pose an unbearable financial burden to the household. Recent in vitro studies demonstrated that beta-lactams combined with rifampicin and clarithromycin are synergistic against M. ulcerans. Consequently, inclusion of amoxicillin/clavulanate in a triple oral therapy may potentially improve and shorten the healing process. The BLMs4BU trial aims to assess whether co-administration of amoxicillin/clavulanate with rifampicin and clarithromycin could reduce BU treatment from 8 to 4 weeks. METHODS: We propose a randomized, controlled, open-label, parallel-group, non-inferiority phase II, multi-centre trial in Benin with participants stratified according to BU category lesions and randomized to two oral regimens: (i) Standard: rifampicin plus clarithromycin therapy for 8 weeks; and (ii) Investigational: standard plus amoxicillin/clavulanate for 4 weeks. The primary efficacy outcome will be lesion healing without recurrence and without excision surgery 12 months after start of treatment (i.e. cure rate). Seventy clinically diagnosed BU patients will be recruited per arm. Patients will be followed up over 12 months and managed according to standard clinical care procedures. Decision for excision surgery will be delayed to 14 weeks after start of treatment. Two sub-studies will also be performed: a pharmacokinetic and a microbiology study. DISCUSSION: If successful, this study will create a new paradigm for BU treatment, which could inform World Health Organization policy and practice. A shortened, highly effective, all-oral regimen will improve care of BU patients and will lead to a decrease in hospitalization-related expenses and indirect and social costs and improve treatment adherence. This trial may also provide information on treatment shortening strategies for other mycobacterial infections (tuberculosis, leprosy, or non-tuberculous mycobacteria infections). TRIAL REGISTRATION: ClinicalTrials.gov NCT05169554 . Registered on 27 December 2021.

The Veterinary Anti-Parasitic Selamectin Is a Novel Inhibitor of the Mycobacterium tuberculosis DprE1 Enzyme.

Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.

Repurposing Avermectins and Milbemycins against Mycobacteroides abscessus and Other Nontuberculous Mycobacteria.

Infections caused by nontuberculous mycobacteria (NTM) are increasing worldwide, resulting in a new global health concern. NTM treatment is complex and requires combinations of several drugs for lengthy periods. In spite of this, NTM disease is often associated with poor treatment outcomes. The anti-parasitic family of macrocyclic lactones (ML) (divided in two subfamilies: avermectins and milbemycins) was previously described as having activity against mycobacteria, including Mycobacterium tuberculosis, Mycobacterium ulcerans, and Mycobacterium marinum, among others. Here, we aimed to characterize the in vitro anti-mycobacterial activity of ML against a wide range of NTM species, including Mycobacteroides abscessus. For this, Minimum Inhibitory Concentration (MIC) values of eight ML were determined against 80 strains belonging to nine different NTM species. Macrocyclic lactones showed variable ranges of anti-mycobacterial activity that were compound and species-dependent. Milbemycin oxime was the most active compound, displaying broad-spectrum activity with MIC lower than 8 mg/L. Time kill assays confirmed MIC data and showed bactericidal and sterilizing activity of some compounds. Macrocyclic lactones are available in many formulations and have been extensively used in veterinary and human medicine with suitable pharmacokinetics and safety properties. This information could be exploited to explore repurposing of anti-helminthics for NTM therapy.

The FICI paradigm: Correcting flaws in antimicrobial in vitro synergy screens at their inception.

Antibiotics have become the corner stone of modern medicine. However, our society is currently facing one of the greatest challenges of its time: the emergence of antimicrobial resistance. It is estimated that if no new therapies are implemented by 2050, 10 million people will die worldwide every year as a result of infections caused by bacteria resistant to current antibiotics; new antimicrobials are thus urgently needed. However, drug development is a tedious and very costly endeavor of hundreds of millions that can take up to 15-20 years from the bench discovery to the bedside. Under this scenario, drug repurposing, which consists in identifying new uses for old, clinically approved drugs, has gathered momentum within the pharmaceutical industry. Because most of these drugs have safety and toxicity information packages available, clinical evaluation could be done in a much shorter period than standard timelines. Synergistic combinations of these clinically approved drugs could also be a promising approach to identify novel antimicrobial therapies that might provide rational choices of available drugs to shorten treatment, increase efficacy, reduce toxicity, prevent resistance and treat infections caused by drug-resistant strains. However, although simple in its conception, translating results from in vitro synergy screens into in vivo efficacy or the clinical practice has proven to be a paramount challenge. In this Commentary, we will discuss common flaws at the inception of synergy research programs, with a special focus on the use of the Fractional Inhibitory Concentration Index (FICI), and evaluate potential interventions that can be made at different developmental pre-clinical stages in order to improve the odds of translation from in vitro studies.

Triple oral beta-lactam containing therapy for Buruli ulcer treatment shortening.

The potential use of clinically approved beta-lactams for Buruli ulcer (BU) treatment was investigated with representative classes analyzed in vitro for activity against Mycobacterium ulcerans. Beta-lactams tested were effective alone and displayed a strong synergistic profile in combination with antibiotics currently used to treat BU, i.e. rifampicin and clarithromycin; this activity was further potentiated in the presence of the beta-lactamase inhibitor clavulanate. In addition, quadruple combinations of rifampicin, clarithromycin, clavulanate and beta-lactams resulted in multiplicative reductions in their minimal inhibitory concentration (MIC) values. The MIC of amoxicillin against a panel of clinical isolates decreased more than 200-fold within this quadruple combination. Amoxicillin/clavulanate formulations are readily available with clinical pedigree, low toxicity, and orally and pediatric available; thus, supporting its potential inclusion as a new anti-BU drug in current combination therapies.

Repurposing clinically approved cephalosporins for tuberculosis therapy.

While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy, and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.

Selamectin Is the Avermectin with the Best Potential for Buruli Ulcer Treatment.

A comprehensive analysis was done to evaluate the potential use of anti-parasitic macrocyclic lactones (including avermectins and milbemycins) for Buruli ulcer (BU) therapy. A panel containing nearly all macrocyclic lactones used in human or in veterinary medicine was analyzed for activity in vitro against clinical isolates of Mycobacterium ulcerans. Milbemycin oxime and selamectin were the most active drugs against M. ulcerans with MIC values from 2 to 8 µg/mL and 2 to 4 µg/mL, respectively. In contrast, ivermectin and moxidectin, which are both in clinical use, showed no significant activity (MIC> 32 µg/mL). Time-kill kinetic assays showed bactericidal activity of selamectin and in vitro pharmacodynamic studies demonstrated exposure-dependent activity. These data together with analyses of published pharmacokinetic information strongly suggest that selamectin is the most promising macrocyclic lactone for BU treatment.

The mycobacterial P55 efflux pump is required for optimal growth on cholesterol.

Cholesterol catabolism is thought to be a key factor contributing to the pathogenesis of Mycobacterium tuberculosis. Previous epistasis and mutant screening studies predicted that the P55 efflux pump (Rv1410c) positively interacts with the Mce4 transporter, a major cholesterol import system of M. tuberculosis and is needed for optimal growth in vitro, in macrophages, and in vivo. Using a combination of cell growth kinetic techniques, cholesterol consumption, and [4-(14)C]cholesterol uptake studies, we demonstrated that the Mycobacterium bovis BCG rv1410c gene indeed is needed for optimal in vitro growth on cholesterol and other carbon sources. Our data, together with previous predictions, support hypotheses that the P55 efflux pump functions in maintaining general metabolism or as a subunit of the Mce4 transport apparatus (catalyzing its assembly or providing cell wall integrity) to allow more efficient cholesterol uptake.

Challenges faced by multidisplinary new investigators on addressing grand challenges in global health.

BACKGROUND: The grand challenges approach aims to spark innovative and transformative strategies to overcome barriers to significant global health issues. Grand Challenges Canada endorses an 'Integrated Innovation™' approach that focuses on the intersection of scientific/technological, social and business innovation. In this article we explore themes emerging from a dialogue between the authors, who are multidisciplinary recipients of the 'Rising Stars in Global Health' award from Grand Challenges Canada, regarding benefits of engaging in integrated innovation research, and recommendations for how this approach may develop in the future. DISCUSSION: Our dialogue followed a semi-structured interview format that addressed three topics: 1) reflections on applying an Integrated Innovation™ approach for global health; 2) thoughts on participation in the Grand Challenges 2012 meeting; and 3) authors' visions of Grand Challenges Canada and the Grand Challenge movement towards 2020. The dialogue was transcribed verbatim and we used thematic analysis techniques to identify, analyze and report themes in the data. Benefits of working using the Grand Challenges approach centered on two themes: a) the potential for scientific breakthrough and b) building interdisciplinary collaborations and a community of scholars. Challenges and opportunities for Grand Challenges in moving forward included: a) capacity building, particularly regarding Integrated Innovation™ and scale-up planning; b) interdisciplinary and international mentorship for new investigators; and c) potential for future commercialization. CONCLUSIONS: Our discussion highlighted that Integrated Innovation™ offers the opportunity to develop new theories, methods and approaches to global health while simultaneously fostering a collaborative spirit grounded in international, interdisciplinary collaborations. However, the arguable over-emphasis on corporatization poses a major challenge for new investigators. We propose a more balanced way forward that can harness technology to foster mentorship across time and space to support the development of such skills and ideas among new investigators.

WhiB7, an Fe-S-dependent transcription factor that activates species-specific repertoires of drug resistance determinants in actinobacteria.

WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.

Targeting Mycobacterium tuberculosis and other microbial pathogens using improved synthetic antibacterial peptides.

The lack of effective therapies for treating tuberculosis (TB) is a global health problem. While Mycobacterium tuberculosis is notoriously resistant to most available antibiotics, we identified synthetic short cationic antimicrobial peptides that were active at low micromolar concentrations (less than 10 µM). These small peptides (averaging 10 amino acids) had remarkably broad spectra of antimicrobial activities against both bacterial and fungal pathogens and an indication of low cytotoxicity. In addition, their antimicrobial activities displayed various degrees of species specificity that were not related to taxonomy. For example, Candida albicans and Staphylococcus aureus were the best surrogates to predict peptide activity against M. tuberculosis, while Mycobacterium smegmatis was a poor surrogate. Principle component analysis of activity spectrum profiles identified unique features associated with activity against M. tuberculosis that reflect their distinctive amino acid composition; active peptides were more hydrophobic and cationic, reflecting increased tryptophan with compensating decreases in valine and other uncharged amino acids and increased lysine. These studies provide foundations for development of cationic antimicrobial peptides as potential new therapeutic agents for TB treatment.