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Aims: The current treatment for Buruli ulcer is based on empirical evidence of efficacy. However, there is an opportunity for shortening its duration and improving response rates. Evolving understanding of the pharmacokinetic-pharmacodynamic relationships provides the basis for a stronger dose rationale for antibiotics. In conjunction with modelling and simulation, it is possible to identify dosing regimens with the highest probability of target attainment (PTA). This investigation aims to: (i) assess the dose rationale for a new combination therapy including amoxicillin/clavulanic acid (AMX/CLV) currently in clinical trials; and (ii) compare its performance with alternative dosing regimens including rifampicin, clarithromycin and AMX/CLV., Methods: In vitro estimates of the minimum inhibitory (MIC) concentration were selected as a measure of the antibacterial activity of different drug combinations. Clinical trial simulations were used to characterize the concentration vs. time profiles of rifampicin, clarithromycin and amoxicillin in a virtual cohort of adult and paediatric patients, considering the effect of baseline covariates on disposition parameters and interindividual variability in exposure. The PTA of each regimen was then assessed using different thresholds of the time above MIC., Results: A weight-banded dosing regimen including 150-600 mg rifampicin once daily, 250-1000 mg clarithromycin and AMX/CLV 22.5 mg/kg /1000 mg twice daily ensures higher PTA than the standard of care with AMX/CLV 45 mg/kg/2000 mg once daily., Conclusion: The higher PTA values support the proposed 4-drug combination (rifampicin, clarithromycin, AMX/CLV) currently under clinical investigation. Our findings also suggest that higher rifampicin doses might contribute to enhanced treatment efficacy., (© 2024 The Author(s). British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Tuberculosis (TB) is historically the world's deadliest infectious disease. New TB drugs that can avoid pre-existing resistance are desperately needed. The β-lactams are the oldest and most widely used class of antibiotics to treat bacterial infections but, for a variety of reasons, they were largely ignored until recently as a potential treatment option for TB. Recently, a growing body of evidence indicates that later-generation carbapenems in the presence of β-lactamase inhibitors could play a role in TB treatment. However, most of these drugs can only be administered intravenously in the clinic. We performed a screening of β-lactams against intracellular Mycobacterium tuberculosis ( Mtb ) and identified sanfetrinem cilexetil as a promising oral β-lactam candidate. Preclinical in vitro and in vivo studies demonstrated that: (i) media composition impacts the activity of sanfetrinem against Mtb , being more potent in the presence of physiologically relevant cholesterol as the only carbon source, compared to the standard broth media; (ii) sanfetrinem shows broad spectrum activity against Mtb clinical isolates, including MDR/XDR strains; (iii) sanfetrinem is rapidly bactericidal in vitro against Mtb despite being poorly stable in the assay media; (iv) there are strong in vitro synergistic interactions with amoxicillin, ethambutol, rifampicin and rifapentine and, (v) sanfetrinem cilexetil is active in an in vivo model of infection. These data, together with robust pre-clinical and clinical studies of broad-spectrum carbapenem antibiotics carried out in the 1990s by GSK, identified sanfetrinem as having potential for treating TB and catalyzed a repurposing proof-of-concept Phase 2a clinical study (NCT05388448) currently underway in South Africa., Competing Interests: Competing interests. RGdR, MPAC, JR, SA, MCI, EPdF, EPH, ASV, AML, SFB, DBA and RHB are or were employees of GSK, a producer of the generic drug amoxicillin/clavulanate and sanfetrinem. SRG, RGdR, AML, DBA and RHB are inventors of the patent describing the antituberculosis activity of sanfetrinem (WO2018206466A1). CJT declare no conflicts of interest. All authors approved the submission of the document.

Aims: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary disease. Mycobacterium abscessus is associated with chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date, no single drug has been developed targeting M. abscessus specifically. The objective of this study was to characterize VOMG, a pyrithione-core drug-like small molecule, as a new compound active against M. abscessus and other pathogens., Methods: A multi-disciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures was used to validate VOMG as a promising anti-M. abscessus drug candidate., Results: To the authors' knowledge, this is the first study to report the in-vitro and in-vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacological (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme., Conclusions: VOMG is a new drug-like molecule active against M. abscessus, inhibiting cell division with broad-spectrum activity against other microbial pathogens., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sáez-López et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2023. Springer Nature Limited.)

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., Competing Interests: The authors declare no competing interests., (© 2023 The Authors.)

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 β-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 β-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 β-lactams against Mkn . A selection of 32 agents including all β-lactam chemical classes (penicillins, cephalosporins, carbapenems and monobactams) with three β-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 β-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.

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., (© 2022. The Author(s).)

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-β-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 14 C-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.

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.

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., Competing Interests: RGdR and AML are employees of GlaxoSmithKline, a producer of the generic drug amoxicillin/clavulanate. All other authors declare no conflicts of interest.

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., Competing Interests: R.G.d.R., A.S.V., F.C., D.B., L.B., A.M.-L. and S.F.-B. are employees of GlaxoSmithKline, a producer of the generic drug amoxicillin/clavulanate (Augmentin). All other authors declare no conflicts of interest. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

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.

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.

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.

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.

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.

Avermectins are a family of macrolides known for their anthelmintic activities and traditionally believed to be inactive against all bacteria. Here we report that members of the family, ivermectin, selamectin, and moxidectin, are bactericidal against mycobacterial species, including multidrug-resistant and extensively drug-resistant clinical strains of Mycobacterium tuberculosis. Avermectins are approved for clinical and veterinary uses and have documented pharmacokinetic and safety profiles. We suggest that avermectins could be repurposed for tuberculosis treatment.

Four butenolides, ramariolides A-D (1-4), have been isolated from the fruiting bodies of the coral mushroom Ramaria cystidiophora. Their structures were elucidated by analysis of 1D and 2D NMR data and a single-crystal X-ray diffraction analysis of 1, and their absolute configurations were established using Mosher's method. The major metabolite, ramariolide A (1), which contains an unusual spiro oxiranebutenolide moiety, showed in vitro antimicrobial activity against Mycobacterium smegmatis and Mycobacterium tuberculosis.

Current tuberculosis treatment regimens are notoriously limited, lengthy and becoming increasingly ineffective due to the emergence of drug-resistant mutant strains of Mycobacterium tuberculosis. The intrinsic resistance of M. tuberculosis to the majority of available drugs relies both on the impermeability of its cell envelope, and its ability to activate specific genes and physiological states. WhiB7 is a transcriptional regulatory protein underlying this adaptive process. Transcription of the whiB7 gene is upregulated in response to a variety of antibiotics having different structures and targets, as well as in response to metabolic signals. The whiB7 regulon activates various systems of intrinsic drug resistance involving antibiotic export, antibiotic inactivation (by chemical modifications of the drug or its target) and significant changes to thiol redox balance. Drugs have been identified that inactivate resistance determinants in the whiB7 regulon, thereby potentiating the activities of diverse antibiotics against M. tuberculosis.

Efflux pumps extrude a wide variety of chemically unrelated compounds conferring multidrug resistance and participating in numerous physiological processes. Mycobacterium tuberculosis possesses many efflux pumps, and their roles in drug resistance and physiology are actively investigated. In this work we found that tap mutant cells showed changes in morphology and a progressive loss of viability upon subcultivation in liquid medium. Transcriptome analysis in Mycobacterium bovis BCG revealed that disruption of the Rv1258c gene, encoding the Tap efflux pump, led to an extensive change in gene expression patterns during stationary phase, with no changes during exponential growth. In stationary phase, Tap inactivation triggered a general stress response and led to a general repression of genes involved in cell wall biosynthesis, in particular the formation of the peptidoglycan; this suggested the accumulation of an unknown Tap substrate that reaches toxic concentrations during stationary phase. We also found that both disruption and overexpression of tap altered susceptibility to many clinically approved antibiotics in M. bovis BCG. Acriflavine and tetracycline accumulation assays and carbonyl cyanide m-chlorophenylhydrazone (CCCP) potentiation experiments demonstrated that this phenotype was due to an active efflux mechanism. These findings emphasize the important role of the Tap efflux pump in bacterial physiology and intrinsic drug resistance.

Intrinsic drug resistance in Mycobacterium tuberculosis limits therapeutic options for treating tuberculosis. The mycobacterial transcriptional regulator whiB7 contributes to intrinsic resistance by activating its own expression and many drug resistance genes in response to antibiotics. To investigate whiB7 activation, we constructed a GFP reporter to monitor its expression, and we used it to investigate the whiB7 promoter and to screen our custom library of almost 600 bioactive compounds, including the majority of clinical antibiotics. Results showed whiB7 was transcribed from a promoter that was conserved across mycobacteria and other actinomycetes, including an AT-rich sequence that was likely targeted by WhiB7. Expression was induced by compounds having diverse structures and targets, independent of the ability of whiB7 to mediate resistance, and was dependent on media composition. Pretreatment with whiB7 activators resulted in clinically relevant increases in intrinsic drug resistance. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced cytoplasm reflected by the ratio of reduced/oxidized mycothiol. These data provided evidence that intrinsic resistance resulting from whiB7 activation is linked to fundamental changes in cell metabolism.

Therapeutic options for tuberculosis (TB) are limited and notoriously ineffective despite the wide variety of potent antibiotics available for treating other bacterial infections. We investigated an approach that enables an expansion of TB therapeutic strategies by using synergistic combinations of drugs. To achieve this, we devised a high-throughput synergy screen (HTSS) of chemical libraries having known pharmaceutical properties, including thousands that are clinically approved. Spectinomycin was used to test the concept that clinically available antibiotics with limited efficacy against Mycobacterium tuberculosis might be used for TB treatment when coadministered with a synergistic partner compound used as a sensitizer. Screens using Mycobacterium smegmatis revealed many compounds in our libraries that acted synergistically with spectinomycin. Among them, several families of antimicrobial compounds, including macrolides and azoles, were also synergistic against M. tuberculosis in vitro and in a macrophage model of M. tuberculosis infection. Strikingly, each sensitizer identified for synergy with spectinomycin uniquely enhanced the activities of other clinically used antibiotics, revealing a remarkable number of unexplored synergistic drug combinations. HTSS also revealed a novel activity for bromperidol, a butyrophenone used as an antipsychotic drug, which was discovered to be bactericidal and greatly enhanced the activities of several antibiotics and drug combinations against M. tuberculosis. Our results suggest that many compounds in the currently available pharmacopoeia could be readily mobilized for TB treatment, including disease caused by multi- and extensively drug-resistant strains for which there are no effective therapies.

Bacterial efflux pumps have traditionally been studied as low-level drug resistance determinants. Recent insights have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion may be a secondary function. In Mycobacterium tuberculosis, little is known about the physiological or drug resistance roles of efflux pumps. Using Mycobacterium bovis BCG as a model system, we showed that deletion of the Rv1410c gene encoding the P55 efflux pump made the strain more susceptible to a range of toxic compounds, including rifampin (rifampicin) and clofazimine, which are first- and second-line antituberculosis drugs. The efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin inhibited the P55-determined drug resistance, suggesting the active export of the compounds by use of the transmembrane proton and electrochemical gradients as sources of energy. In addition, the P55 efflux pump mutant was more susceptible to redox compounds and displayed increased intracellular redox potential, suggesting an essential role of the efflux pump in detoxification processes coupled to oxidative balance within the cell. Finally, cells that lacked the p55 gene displayed smaller colony sizes and had a growth defect in liquid culture. This, together with an increased susceptibility to the cell wall-targeting compounds bacitracin and vancomycin, suggested that P55 is needed for proper cell wall assembly and normal growth in vitro. Thus, P55 plays a fundamental role in oxidative stress responses and in vitro cell growth, in addition to contributing to intrinsic antibiotic resistance. Inhibitors of the P55 efflux pump could help to improve current treatments for tuberculosis.

Objectives: To characterize the efflux pump encoded by the gene Rv2333c from Mycobacterium tuberculosis, and assess its contribution to intrinsic antibiotic resistance using Mycobacterium bovis BCG as a model organism., Methods: Firstly, the Rv2333c gene was expressed from a multicopy plasmid in M. bovis BCG. Secondly, the gene was inactivated in the chromosome of M. bovis BCG. Antibiotic susceptibility tests and tetracycline uptake/efflux experiments were carried out with the strains mentioned above., Results: When the Rv2333c gene was inactivated in the M. bovis BCG chromosome, there was a decrease in the MIC values of spectinomycin and tetracycline, and an increase in [3H]tetracycline accumulation. When the Rv2333c gene was cloned into a multicopy plasmid, there was an increase in the MIC values of spectinomycin and tetracycline, and a decrease in [3H]tetracycline accumulation. These results indicate that both antibiotics are substrates of the Rv2333c efflux pump, which has been named Stp, for Spectinomycin Tetracycline efflux Pump., Conclusions: The Rv2333c efflux pump (Stp protein) of M. tuberculosis contributes to intrinsic spectinomycin and tetracycline resistance.

We have isolated the aph(3")-Ic gene, encoding an aminoglycoside 3"-O-phosphotransferase [APH(3")-Ic], from a genomic library of an environmental Mycobacterium fortuitum strain, selecting for streptomycin resistance. APH(3")-Ic phosphorylates and inactivates streptomycin. Similar genes have been described in Streptomyces griseus and plasmid RSF1010. It is also present in some M. fortuitum clinical isolates.

Objectives: The aim of this study was to characterize the efflux pump Tap from Mycobacterium fortuitum, to test its sensitivity to well known efflux inhibitors, to study the interaction between tetracycline and these compounds and to test the ability of these compounds to overcome efflux pump-mediated tetracycline resistance. For all these studies, we produced Tap protein in Mycobacterium smegmatis., Methods: Antibiotic susceptibility tests, tetracycline uptake/efflux experiments and checkerboard synergy tests., Results: Tetracycline uptake/efflux experiments showed that Tap protein from M. fortuitum uses the electrochemical gradient across the cytoplasmic membrane to extrude tetracycline from the cell. This efflux activity is inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine, consistent with the decrease in MIC observed in antibiotic susceptibility testing in the presence of these inhibitors. Accumulation was not inhibited in experiments in which o-vanadate and chlorpromazine (CPZ) were tested. Inhibitor-treated cells used glycerol as a carbon source to re-establish the electrochemical gradient across the membrane and to restore efflux activity. CCCP, reserpine and CPZ reduced the MIC of tetracycline in the M. smegmatis strain expressing the Tap protein, whereas o-vanadate increased the MIC. We also observed synergy between tetracycline and CPZ or reserpine, and antagonism with o-vanadate., Conclusions: The Tapfor efflux pump uses the electrochemical gradient to extrude tetracycline from the cell. This efflux activity can be inhibited by several compounds. This suggests that similar compounds could be used to overcome antibiotic resistance mediated by efflux pumps.