Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status.

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

High-throughput functional genomics: A (myco)bacterial perspective.

Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high-throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function. Here, we summarize the evolution of bacterial high-throughput functional genomics, focusing primarily on transposon (Tn)-based mutagenesis and the construction of arrayed mutant libraries in diverse bacterial systems. We also consider the contributions of CRISPR interference as a transformative technique for probing bacterial gene function at scale. Throughout, we situate our analysis within the context of functional genomics of mycobacteria, focusing specifically on the potential to yield insights into M. tuberculosis pathogenicity and vulnerabilities for new drug and regimen development. Finally, we offer suggestions for future approaches that might be usefully applied in elucidating the complex cellular biology of this major human pathogen.

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition.

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.

Capture and visualization of live Mycobacterium tuberculosis bacilli from tuberculosis patient bioaerosols.

Interrupting transmission is an attractive anti-tuberculosis (TB) strategy but it remains underexplored owing to our poor understanding of the events surrounding transfer of Mycobacterium tuberculosis (Mtb) between hosts. Determining when live, infectious Mtb bacilli are released and by whom has proven especially challenging. Consequently, transmission chains are inferred only retrospectively, when new cases are diagnosed. This process, which relies on molecular analyses of Mtb isolates for epidemiological fingerprinting, is confounded by the prolonged infectious period of TB and the potential for transmission from transient exposures. We developed a Respiratory Aerosol Sampling Chamber (RASC) equipped with high-efficiency filtration and sampling technologies for liquid-capture of all particulate matter (including Mtb) released during respiration and non-induced cough. Combining the mycobacterial cell wall probe, DMN-trehalose, with fluorescence microscopy of RASC-captured bioaerosols, we detected and quantified putative live Mtb bacilli in bioaerosol samples arrayed in nanowell devices. The RASC enabled non-invasive capture and isolation of viable Mtb from bioaerosol within 24 hours of collection. A median 14 live Mtb bacilli (range 0-36) were isolated in single-cell format from 90% of confirmed TB patients following 60 minutes bioaerosol sampling. This represented a significant increase over previous estimates of transmission potential, implying that many more organisms might be released daily than commonly assumed. Moreover, variations in DMN-trehalose incorporation profiles suggested metabolic heterogeneity in aerosolized Mtb. Finally, preliminary analyses indicated the capacity for serial image capture and analysis of nanowell-arrayed bacilli for periods extending into weeks. These observations support the application of this technology to longstanding questions in TB transmission including the propensity for asymptomatic transmission, the impact of TB treatment on Mtb bioaerosol release, and the physiological state of aerosolized bacilli.

Exploration of 4-aminopyrrolo[2,3-d]pyrimidine as antitubercular agents.

Tuberculosis (TB) is one of the leading causes of death worldwide. Developing new anti-TB compounds using cost-effective processes is critical to reduce TB incidence and accomplish the End TB Strategy milestone. Herein, we describe the synthesis and structure-activity relationships of a library of thirty 7H-Pyrrolo[2,3-d]pyrimidine derivatives providing insights into the contributions of different aromatic, aryl and alkyl substitution at the C-4 position of the 7-deazapurine ring. The minimum inhibitory concentration (MIC) of the compounds against the green fluorescent protein (GFP) reporter strain of Mycobacterium tuberculosis was assayed using the standard broth microdilution method, and cell toxicity was determined using the MTT assay. Sixteen compounds displayed in vitro activity against the GFP reporter strain of Mycobacterium tuberculosis with MIC90 values of 0.488-62.5 µM. This study highlights the most potent derivative, N-(4-phenoxy phenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine with a MIC90 value of 0.488 µM and was non-cytotoxic to the Vero cell line. Moreover, all the potent compounds from this series have a ClogP value less than 4 and molecular weight < 400; thus, likely to maintain drug-likeness during lead optimisation.

Aerosolization of Mycobacterium tuberculosis by Tidal Breathing.

Rationale: Interrupting tuberculosis (TB) transmission requires an improved understanding of how and when the causative organism, Mycobacterium tuberculosis (Mtb), is aerosolized. Although cough is commonly assumed to be the dominant source of Mtb aerosols, recent evidence of cough-independent Mtb release implies the contribution of alternative mechanisms. Objectives: To compare the aerosolization of Mtb bacilli and total particulate matter from patients with TB during three separate respiratory maneuvers: tidal breathing (TiBr), FVC, and cough. Methods: Bioaerosol sampling and Mtb enumeration by live-cell, fluorescence microscopy were combined with real-time measurement of CO2 concentration and total particle counts from 38 patients with GeneXpert-positive TB before treatment initiation. Measurements and Main Results: For all maneuvers, the proportions of particles detected across five size categories were similar, with most particles falling between 0.5-5 µm. Although total particle counts were 4.8-fold greater in cough samples than either TiBr or FVC, all three maneuvers returned similar rates of positivity for Mtb. No correlation was observed between total particle production and Mtb count. Instead, for total Mtb counts, the variability between individuals was greater than the variability between sampling maneuvers. Finally, when modelled using 24-hour breath and cough frequencies, our data indicate that TiBr might contribute more than 90% of the daily aerosolized Mtb among symptomatic patients with TB. Conclusions: Assuming the number of viable Mtb organisms released offers a reliable proxy of patient infectiousness, our observations imply that TiBr and interindividual variability in Mtb release might be significant contributors to TB transmission among active cases.

Molecular Detection of Airborne Mycobacterium tuberculosis in South African High Schools.

Rationale: South African adolescents carry a high tuberculosis disease burden. It is not known if schools are high-risk settings for Mycobacterium tuberculosis (MTB) transmission. Objectives: To detect airborne MTB genomic DNA in classrooms. Methods: We studied 72 classrooms occupied by 2,262 students in two South African schools. High-volume air filtration was performed for median 40 (interquartile range [IQR], 35-54) minutes and assayed by droplet digital PCR (ddPCR)-targeting MTB region of difference 9 (RD9), with concurrent CO2 concentration measurement. Classroom data were benchmarked against public health clinics. Students who consented to individual tuberculosis screening completed a questionnaire and sputum collection (Xpert MTB/RIF Ultra) if symptom positive. Poisson statistics were used for MTB RD9 copy quantification. Measurements and Main Results: ddPCR assays were positive in 13/72 (18.1%) classrooms and 4/39 (10.3%) clinic measurements (P = 0.276). Median ambient CO2 concentration was 886 (IQR, 747-1223) ppm in classrooms versus 490 (IQR, 405-587) ppm in clinics (P < 0.001). Average airborne concentration of MTB RD9 was 3.61 copies per 180,000 liters in classrooms versus 1.74 copies per 180,000 liters in clinics (P = 0.280). Across all classrooms, the average risk of an occupant inhaling one MTB RD9 copy was estimated as 0.71% during one standard lesson of 35 minutes. Among 1,836/2,262 (81.2%) students who consented to screening, 21/90 (23.3%) symptomatic students produced a sputum sample, of which one was Xpert MTB/RIF Ultra positive. Conclusions: Airborne MTB genomic DNA was detected frequently in high school classrooms. Instantaneous risk of classroom exposure was similar to the risk in public health clinics.

Estimating Tuberculosis Transmission Risks in a Primary Care Clinic in South Africa: Modeling of Environmental and Clinical Data.

BACKGROUND: Congregate settings, such as healthcare clinics, may play an essential role in Mycobacterium tuberculosis (Mtb) transmission. Using patient and environmental data, we studied transmission at a primary care clinic in South Africa. METHODS: We collected patient movements, cough frequency, and clinical data, and measured indoor carbon dioxide (CO2) levels, relative humidity, and Mtb genomes in the air. We used negative binomial regression model to investigate associations. RESULTS: We analyzed 978 unique patients who contributed 14 795 data points. The median patient age was 33 (interquartile range [IQR], 26-41) years, and 757 (77.4%) were female. Overall, median CO2 levels were 564 (IQR 495-646) parts per million and were highest in the morning. Median number of coughs per day was 466 (IQR, 368-503), and overall median Mtb DNA copies/µL/day was 4.2 (IQR, 1.2-9.5). We found an increased presence of Mtb DNA in the air of 32% (95% credible interval, 7%-63%) per 100 additional young adults (aged 15-29 years) and 1% (0-2%) more Mtb DNA per 10% increase of relative humidity. Estimated cumulative transmission risks for patients attending the clinic monthly for at least 1 hour range between 9% and 29%. CONCLUSIONS: We identified young adults and relative humidity as potentially important factors for transmission risks in healthcare clinics. Our approach should be used to detect transmission and evaluate infection control interventions.

Nitrothiazole-Thiazolidinone Hybrids: Synthesis and in Vitro Antimicrobial Evaluation.

Herein we report the synthesis of novel compounds inspired by the antimicrobial activities of nitroazole and thiazolidin-4-one based compounds reported in the literature. Target compounds were investigated in vitro for antitubercular, antibacterial, antifungal, and overt cell toxicity properties. All compounds exhibited potent antitubercular activity. Most compounds exhibited low micromolar activity against S. aureus and C. albicans with no overt cell toxicity against HEK-293 cells nor haemolysis against human red blood cells. Notably, compound 3b exhibited low to sub-micromolar activities against Mtb, MRSA, and C. albicans. 3b showed superior activity (0.25 µg/ml) against MRSA compared to vancomycin (1 µg/ml).

Investigation of quinolone-tethered aminoguanidine as novel antibacterial agents.

A recent study identified quinolone-based thiosemicarbazone with an MIC90 value of 2 µM against Mycobacterium tuberculosis (Mtb). Herein, we report further optimization of the previous hit, which led to the discovery of quinolone-tethered aminoguanidine molecules with generally good antitubercular activity. Compounds 7f and 8e emerged as the hits of the series with submicromolar antitubercular activity, exhibiting MIC90 values of 0.49/0.90 and 0.49/0.60 µM, respectively, in the 7H9 CAS GLU Tx medium. This shows a fivefold increase in antitubercular activity compared to the previous study. Target compounds were also screened against ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. However, the series generally exhibited poor antibacterial activities, with only compounds 8d and 8e demonstrating >50% growth inhibition of Staphylococcus aureus and Pseudomonas aeruginosa at 32 µg/ml. The compounds displayed selective antitubercular activity as they showed no cytotoxicity effects against two noncancerous human cell lines. In silico studies predict 7f to have good solubility, no inhibitory effect on cytochrome P450 isoenzymes, and to be a non-pan-assay interfering compound.

De Novo Cobalamin Biosynthesis, Transport, and Assimilation and Cobalamin-Mediated Regulation of Methionine Biosynthesis in Mycobacterium smegmatis.

Cobalamin is an essential cofactor in all domains of life, yet its biosynthesis is restricted to some bacteria and archaea. Mycobacterium smegmatis, an environmental saprophyte frequently used as surrogate for the obligate human pathogen M. tuberculosis, carries approximately 30 genes predicted to be involved in de novo cobalamin biosynthesis. M. smegmatis also encodes multiple cobalamin-dependent enzymes, including MetH, a methionine synthase that catalyzes the final reaction in methionine biosynthesis. In addition to metH, M. smegmatis possesses a cobalamin-independent methionine synthase, metE, suggesting that enzyme use-MetH versus MetE-is regulated by cobalamin availability. Consistent with this notion, we previously described a cobalamin-sensing riboswitch controlling metE expression in M. tuberculosis Here, we apply a targeted mass spectrometry-based approach to confirm de novo cobalamin biosynthesis in M. smegmatis during aerobic growth in vitro We also demonstrate that M. smegmatis can transport and assimilate exogenous cyanocobalamin (CNCbl; also known as vitamin B12) and its precursor, dicyanocobinamide ([CN]2Cbi). However, the uptake of CNCbl and (CN)2Cbi in this organism is restricted and seems dependent on the conditional essentiality of the cobalamin-dependent methionine synthase. Using gene and protein expression analyses combined with single-cell growth kinetics and live-cell time-lapse microscopy, we show that transcription and translation of metE are strongly attenuated by endogenous cobalamin. These results support the inference that metH essentiality in M. smegmatis results from riboswitch-mediated repression of MetE expression. Moreover, differences observed in cobalamin-dependent metabolism between M. smegmatis and M. tuberculosis provide some insight into the selective pressures which might have shaped mycobacterial metabolism for pathogenicity.IMPORTANCE Alterations in cobalamin-dependent metabolism have marked the evolution of Mycobacterium tuberculosis into a human pathogen. However, the role(s) of cobalamin in mycobacterial physiology remains poorly understood. Using the nonpathogenic saprophyte M. smegmatis, we investigated the production of cobalamin, transport and assimilation of cobalamin precursors, and the role of cobalamin in regulating methionine biosynthesis. We confirm constitutive de novo cobalamin biosynthesis in M. smegmatis, in contrast with M. tuberculosis, which appears to lack de novo cobalamin biosynthetic capacity. We also show that uptake of cyanocobalamin (vitamin B12) and its precursors is restricted in M. smegmatis, apparently depending on the cofactor requirements of the cobalamin-dependent methionine synthase. These observations establish M. smegmatis as an informative foil to elucidate key metabolic adaptations enabling mycobacterial pathogenicity.

Synthesis and comparison of in vitro dual anti-infective activities of novel naphthoquinone hybrids and atovaquone.

A principal factor that contributes towards the failure to eradicate leishmaniasis and tuberculosis infections is the reduced efficacy of existing chemotherapies, owing to a continuous increase in multidrug-resistant strains of the causative pathogens. This accentuates the dire need to develop new and effective drugs against both plights. A series of naphthoquinone-triazole hybrids was synthesized and evaluated in vitro against Leishmania (L.) and Mycobacterium tuberculosis (Mtb) strains. Their cytotoxicities were also evaluated, using the human embryonic kidney cell line (HEK-293). The hybrids were found to be non-toxic towards human cells and had demonstrated micromolar cellular antileishmanial and antimycobacterial potencies. Hybrid 13, i.e. 2-{[1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl]methoxy}naphthalene-1,4-dione was the most active of all. It was found with MIC90 0.5 µM potency against Mtb in a protein free medium, and with half-maxima inhibitory concentrations (IC50) of 0.81 µM and 1.48 µM against the infective promastigote parasites of L. donavani and L. major, respectively, with good selectivity towards these pathogens (SI 22 - 65). Comparatively, the clinical naphthoquinone, atovaquone, although less cytotoxic, was found to be two-fold less antimycobacterial potent, and six- to twelve-fold less active against leishmania. Hybrid 13 may therefore stand as a potential anti-infective hit for further development in the search for new antitubercular and antileishmanial drugs. Elucidation of its exact mechanism of action and molecular targets will constitute future endeavour.

Easy-To-Access Quinolone Derivatives Exhibiting Antibacterial and Anti-Parasitic Activities.

The cell wall of Mycobacterium tuberculosis (Mtb) has a unique structural organisation, comprising a high lipid content mixed with polysaccharides. This makes cell wall a formidable barrier impermeable to hydrophilic agents. In addition, during host infection, Mtb resides in macrophages within avascular necrotic granulomas and cavities, which shield the bacterium from the action of most antibiotics. To overcome these protective barriers, a new class of anti-TB agents exhibiting lipophilic character have been recommended by various reports in literature. Herein, a series of lipophilic heterocyclic quinolone compounds was synthesised and evaluated in vitro against pMSp12::GFP strain of Mtb, two protozoan parasites (Plasmodium falciparum and Trypanosoma brucei brucei) and against ESKAPE pathogens. The resultant compounds exhibited varied anti-Mtb activity with MIC90 values in the range of 0.24-31 µM. Cross-screening against P. falciparum and T.b. brucei, identified several compounds with antiprotozoal activities in the range of 0.4-20 µM. Compounds were generally inactive against ESKAPE pathogens, with only compounds 8c, 8g and 13 exhibiting moderate to poor activity against S. aureus and A. baumannii.

Isolation and characterization of new phenolic siderophores with antimicrobial properties from Pseudomonas sp. UIAU-6B.

Five new phenolic siderophores 1-5 were isolated from the organic extract of a culture broth in a modified SGG medium of Pseudomonas sp. UIAU-6B, obtained from sediments collected from the Oyun river in North Central Nigeria. The structure of the new compounds, pseudomonin A-C (1-3) and pseudomobactin A and B (4 and 5) isolated alongside two known compounds, pseudomonine (6) and salicylic acid (7), were elucidated based on high-resolution mass spectrometry, 1D and 2D NMR analyses. The absolute configuration of the threonine residue in compounds 1-5 was determined by Marfey analysis. The antimicrobial evaluation of compound 4 exhibited the most potent activity against vancomycin-sensitive Enterococcus faecium VS144754, followed by 3 and 5, with MIC values ranging from 8 to 32 µg/mL. Compounds 2 and 3 exhibited moderate activity against Mycobacterium tuberculosis H37Rv, with MIC values of 7.8 and 15.6 µg/mL, respectively. Plausible biosynthetic hypotheses toward the new compounds 1-5 were proposed.

Harnessing Biological Insight to Accelerate Tuberculosis Drug Discovery.

Tuberculosis (TB) is the leading cause of mortality globally resulting from an infectious disease, killing almost 1.6 million people annually and accounting for approximately 30% of deaths attributed to antimicrobial resistance (AMR). This despite the widespread administration of a neonatal vaccine, and the availability of an effective combination drug therapy against the causative agent, Mycobacterium tuberculosis (Mtb). Instead, TB prevalence worldwide is characterized by high-burden regions in which co-epidemics, such as HIV, and social and economic factors, undermine efforts to control TB. These elements additionally ensure conditions that favor the emergence of drug-resistant Mtb strains, which further threaten prospects for future TB control. To address this challenge, significant resources have been invested in developing a TB drug pipeline, an initiative given impetus by the recent regulatory approval of two new anti-TB drugs. However, both drugs have been reserved for drug-resistant disease, and the seeming inevitability of new resistance plus the recognized need to shorten the duration of chemotherapy demands continual replenishment of the pipeline with high-quality "hits" with novel mechanisms of action. This represents a massive challenge, which has been undermined by key gaps in our understanding of Mtb physiology and metabolism, especially during host infection. Whereas drug discovery for other bacterial infections can rely on predictive in vitro assays and animal models, for Mtb, inherent metabolic flexibility and uncertainties about the nutrients available to infecting bacilli in different host (micro)environments instead requires educated predictions or demonstrations of efficacy in animal models of arguable relevance to human disease. Even microbiological methods for enumeration of viable mycobacterial cells are fraught with complication. Our research has focused on elucidating those aspects of mycobacterial metabolism that contribute to the robustness of the bacillus to host immunological defenses and applied antibiotics and that, possibly, drive the emergence of drug resistance. This work has identified a handful of metabolic pathways that appear vulnerable to antibiotic targeting. Those highlighted, here, include the inter-related functions of pantothenate and coenzyme A biosynthesis and recycling and nucleotide metabolism-the last of which reinforces our view that DNA metabolism constitutes an under-explored area for new TB drug development. Although nonessential functions have traditionally been deprioritized for antibiotic development, a common theme emerging from this work is that these very functions might represent attractive targets because of the potential to cripple mechanisms critical to bacillary survival under stress (for example, the RelMtb-dependent stringent response) or to adaptability under unfavorable, potentially lethal, conditions including antibiotic therapy (for example, DnaE2-dependent SOS mutagenesis). The bar, however, is high: demonstrating convincingly the likely efficacy of this strategy will require innovative models of human TB disease. In the concluding section, we focus on the need for improved techniques to elucidate mycobacterial metabolism during infection and its impact on disease outcomes. Here, we argue that developments in other fields suggest the potential to break through this barrier by harnessing chemical-biology approaches in tandem with the most advanced technologies. As researchers based in a high-burden country, we are impelled to continue participating in this important endeavor.

Synthesis and biological evaluation of bis-N2,N2'-(4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccinodihydrazides.

A series of N2,N2'-bis[4-hydroxycoumarin-3-yl)ethylidene]-2,3-dihydroxysuccino-hydrazides, containing 4-hydroxycoumarin, hydrazine and tartaric acid moieties, have been prepared and examined for possible biological activity. Several of these compounds exhibit promising HIV-1 integrase inhibition (IC50 = 3.5 µM), and anti-T. brucei (32% viability) and anti-mycobacterial (Visual MIC90 = 15.63 µM) activity.

Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol.

BACKGROUND: Tuberculosis (TB) is transmitted in bioaerosols containing Mycobacterium tuberculosis (Mtb). Despite being central to ongoing TB transmission, no routine diagnostic assay exists to measure Mtb in bioaerosols. Furthermore, published studies of Mtb in bioaerosol samples have been limited to individuals with sputum-positive pulmonary TB. Notably, TB diagnosis is based on clinical symptoms and sputum laboratory findings. This is despite the fact that approximately half of all patients commencing TB treatment are sputum-negative, resulting in a high proportion of presumptive treatments. Here, we propose to use a sensitive air sampling protocol to investigate the prevalence of Mtb-containing bioaerosols in both sputum-positive and sputum-negative TB suspects, at the same time evaluating the potential to identify unrecognized transmitters of TB. METHODS: Our parallel-group design will identify viable Mtb in bioaerosols produced by individuals attending a TB clinic in South Africa. Sampling will be performed on eligible individuals presenting with symptoms indicative of TB and repeated at 14 days if initially positive. Participants will be prospectively classified into three distinct groups based on National TB Control Program (NTBCP) criteria: Group A, TB notification with sputum-based laboratory confirmation; Group B, TB notification with empiric diagnosis; and Group C, individuals not notified. Group C individuals with detectable Mtb bioaerosol will be monitored until resolution of clinical and laboratory status. Collection of bioaerosol specimens will be via two consecutive sampling modalities: (1) direct sampling following a specific respiratory manoeuvre; and (2) indirect sampling during passive respiratory activity. Bioaerosol specimens will be analyzed for viable Mtb using DMN-trehalose staining and live-cell fluorescence microscopy. Mtb genomes and mycobacterial and host lipids will be detected using droplet digital PCR and mass spectrometry analyses, respectively. The primary objective is to determine the prevalence of Mtb bioaerosols in all TB clinic attendees and in each of the groups. Secondary objectives are to investigate differences in prevalence of Mtb bioaerosol by HIV status and current isoniazid preventive therapy (IPT) use; we will also determine the impact of anti-TB chemotherapy on Mtb-containing bioaerosol production. DISCUSSION: Respiratory bioaerosol has a potential role in non-invasive TB diagnosis, infectivity measurement and treatment monitoring. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04241809 . Date of Registration: 27/1/2020.

Correction to: Bioaerosol sampling of patients with suspected pulmonary tuberculosis: a study protocol.

An amendment to this paper has been published and can be accessed via the original article.