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The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.
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Lipopolisacáridos , Tuberculosis , Humanos , Animales , Ratones , Manosa , InflamaciónRESUMEN
Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and are implicated in the zoonotic transmission of leprosy in the United States. In Mexico, the existence of such a reservoir remains to be characterized. We describe a wild armadillo infected by M. leprae in the state of Nuevo León, Mexico.
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Armadillos , Lepra , Animales , Armadillos/microbiología , Reservorios de Enfermedades/microbiología , Lepra/diagnóstico , Lepra/epidemiología , Lepra/veterinaria , México/epidemiología , Mycobacterium leprae/genéticaRESUMEN
Tuberculosis (TB) remains a major cause of morbidity and mortality, particularly in low- and middle-income countries where access to health care workers, cold-chain storage, and sterile water sources may be limited. Inhaled drug delivery is a promising alternative to systemic delivery of antimycobacterial drugs, as it enables rapid achievement of high infection-site drug concentrations. The off-patent drug clofazimine (CFZ) may be particularly suitable for this route, given its known systemic toxicities. In this study, micronized CFZ particles produced by air jet milling were assessed for shelf-stability, pharmacokinetics, and anti-TB efficacy by the oral and pulmonary routes in BALB/c mice. Intratracheal instillation of micronized CFZ particles produced several-fold higher lung concentrations after a single 30 mg/kg dose compared to delivery via oral gavage, and faster onset of bactericidal activity was observed in lungs of mice with chronic Mycobacterium tuberculosis infection compared to the oral route. Both infection status and administration route affected the multidose pharmacokinetics (PK) of micronized CFZ. Increased lung and spleen accumulation of the drug after pulmonary administration was noted in infected mice compared to naive mice, while the opposite trend was noted in the oral dosing groups. The infection-dependent PK of inhaled micronized CFZ may point to a role of macrophage trafficking in drug distribution, given the intracellular-targeting nature of the formulation. Lastly, air jet milled CFZ exhibited robustness to storage-induced chemical degradation and changes in aerosol performance, thereby indicating the suitability of the formulation for treatment of TB in regions with limited cold chain supply.
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Mycobacterium tuberculosis , Tuberculosis , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Clofazimina/farmacología , Ratones , Ratones Endogámicos BALB C , Tuberculosis/tratamiento farmacológico , AguaRESUMEN
BACKGROUND: Mycobacterium abscessus causes chronic pulmonary infections. Owing to its resistance to most classes of antibiotics, treatment is complex and cure rates are only 45%. Tigecycline is active against M. abscessus, but severe toxicity and the need for IV administration limit its use. OBJECTIVES: To assess the potential of inhaled tigecycline as a treatment for M. abscessus pulmonary disease, by measuring its efficacy in a mouse model of chronic M. abscessus pulmonary disease, establishing the intracellular activity of tigecycline against M. abscessus in human macrophages and measuring the activity of tigecycline in the sputum of cystic fibrosis patients. METHODS: We infected GM-CSF knockout mice with M. abscessus by intrapulmonary aerosol. Infected mice were treated with tigecycline in 0.25, 1.25 and 2.5 mg doses, by inhalation, or untreated, for 28 days. Tigecycline was added to human peripheral blood-derived macrophages infected with M. abscessus to assess its intracellular activity. We performed a time-kill kinetics experiment of tigecycline against M. abscessus with and without sputum of cystic fibrosis patients. RESULTS: Inhaled tigecycline proved highly effective against M. abscessus in GM-CSF knockout mice. The effect was dose dependent. Tigecycline showed potent activity against M. abscessus in macrophages and retained most of its activity in the presence of sputum of cystic fibrosis patients. CONCLUSIONS: Inhaled tigecycline may represent a viable treatment option for M. abscessus pulmonary disease, where treatment outcomes are currently very poor. A stable and safe formulation is required to proceed to further pharmacodynamic studies and ultimately clinical trials.
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Enfermedades Pulmonares , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Animales , Antibacterianos/uso terapéutico , Humanos , Enfermedades Pulmonares/tratamiento farmacológico , Ratones , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , TigeciclinaRESUMEN
The Nix-TB clinical trial evaluated a new 6-month regimen containing three-oral-drugs; bedaquiline (B), pretomanid (Pa) and linezolid (L) (BPaL regimen) for treatment of tuberculosis (TB). This regimen achieved remarkable results as almost 90% of the multidrug resistant (MDR) or extensively drug resistant (XDR) TB participants were cured but many patients also developed severe adverse effects (AEs). The AEs were associated with the long-term administration of the protein synthesis inhibitor linezolid. Spectinamide 1599 (S) is also a protein synthesis inhibitor of Mycobacterium tuberculosis with an excellent safety profile but which lacks oral bioavailability. Here we hypothesize that inhaled spectinamide 1599, combined with BPa --BPaS regimen--has similar efficacy to that of BPaL regimen while simultaneously avoiding the L-associated AEs. The BPaL and BPaS regimens were compared in the Balb/c and C3HeB/FeJ murine chronic TB efficacy models. After 4-weeks of treatment, both regimens promoted equivalent bactericidal effect in both TB murine models. However, treatment with BPaL resulted in significant weight loss and the complete blood count suggested development of anemia. These effects were not similarly observed in mice treated with BPaS. BPaL treatment also decreased myeloid to erythroid ratio and increased concentration of proinflammatory cytokines in bone marrow compared to mice receiving BPaS regimen. During therapy both regimens improved the lung lesion burden, reduced neutrophil and cytotoxic T cells counts while increased the number of B and helper and regulatory T cells. These combined data suggest that inhaled spectinamide 1599 combined with BPa is an effective TB regimen that avoids L-associated AEs. IMPORTANCE: Tuberculosis (TB) is an airborne infectious disease that spreads via aerosols containing Mycobacterium tuberculosis (Mtb), the causative agent of TB. TB can be cured by administration of 3-4 drugs for 6-9 months but there are limited treatment options for patients infected with multidrug (MDR) and extensively resistant (XDR) strains of Mtb. BPaL is a new all-oral combination of drugs consisting of Bedaquiline (B), Pretomanid (Pa) and Linezolid (L). This regimen was able to cure â¼90% of MDR and XDR TB patients in clinical trials but many patients developed severe adverse effects (AEs) associated to the long-term administration of linezolid. We evaluated a new regimen in which Linezolid in the BPaL regimen was replaced with inhaled spectinamide 1599. In the current study, we demonstrate that 4-weeks of treatment with inhaled spectinamide 1599 in combination with Bedaquiline and Pretomanid has equivalent efficacy to the BPaL drug combination and avoids the L-associated-AEs.
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The benzothiazole amide CRS0393 demonstrated excellent in vitro activity against nontuberculous mycobacteria (NTM), including M. abscessus isolates from cystic fibrosis (CF) patients, with minimum inhibitory concentrations (MICs) of ≤0.03-0.5 µg/mL. The essential transport protein MmpL3 was confirmed as the target via analysis of spontaneous resistant mutants and further biological profiling. In mouse pharmacokinetic studies, intratracheal instillation of a single dose of CRS0393 resulted in high concentrations of drug in epithelial lining fluid (ELF) and lung tissue, which remained above the M. abscessus MIC for at least 9 hours post-dose. This exposure resulted in a penetration ratio of 261 for ELF and 54 for lung tissue relative to plasma. CRS0393 showed good oral bioavailability, particularly when formulated in kolliphor oil, with a lung-to-plasma penetration ratio ranging from 0.5 to 4. CRS0393 demonstrated concentration-dependent reduction of intracellular M. abscessus in a THP-1 macrophage infection model. CRS0393 was well tolerated following intranasal administration (8 mg/kg) or oral dosing (25 mg/kg) once daily for 28 days in dexamethasone-treated C3HeB/FeJ mice. Efficacy against M. abscessus strain 103 was achieved via the intranasal route, while oral dosing will need further optimization. CRS0393 holds promise for development as a novel agent with broad antimycobacterial activity.
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Fibrosis Quística , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium tuberculosis , Ratones , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Micobacterias no Tuberculosas , Pulmón , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/microbiología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Pruebas de Sensibilidad MicrobianaRESUMEN
Since 1966, rifampicin (RIF) has been considered one of the most potent drugs in the treatment of tuberculosis (TB), which is caused by infection with M. tuberculosis (Mtb). New nanostructured formulations for RIF delivery and alternative routes of administration have been studied as potential forms of treatment. This study evaluates a liquid crystal system for RIF delivery, using alternative drug delivery routes. The systems developed are composed of surfactant, oleylamine, and soy phosphatidylcholine. With the aid of polarized light microscopy, it was possible to determine that the developed systems had a hexagonal mesophase. All systems developed showed non-Newtonian pseudoplasticity and a high degree of thixotropy. Liquid crystal systems with RIF showed an increase in elastic potential, indicating greater mu-coadhesiveness. The evaluation of mucoadhesive forces revealed an increase in the mucoadhesive potential in the presence of mucus, indicating the presence of satisfactory mucoadhesive forces. The 9DR and 10DR liquid crystal systems, when submitted to Differential Scanning Calorimetry analysis, remained structured even at temperatures above 100 °C, showing excellent stability. The developed liquid crystal systems showed a tolerable degree of cytotoxicity and bactericidal potential, for example, the 9DR system demonstrated a reduction in bacterial load after the third day and reached zero CFU on the seventh day of the test. The developed systems were also evaluated in the preclinical model of Mtb-infected mice, using the nasal, sublingual, and cutaneous route for the delivery of RIF associated with a nanostructured liquid crystal system as a possible tool in the treatment of TB.
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A search for alternative Mycobacterium abscessus treatments led to our interest in the two-component regulator DosRS, which, in Mycobacterium tuberculosis, is required for the bacterium to establish a state of nonreplicating, drug-tolerant persistence in response to a variety of host stresses. We show here that the genetic disruption of dosRS impairs the adaptation of M. abscessus to hypoxia, resulting in decreased bacterial survival after oxygen depletion, reduced tolerance to a number of antibiotics in vitro and in vivo, and the inhibition of biofilm formation. We determined that three antimalarial drugs or drug candidates, artemisinin, OZ277, and OZ439, can target DosS-mediated hypoxic signaling in M. abscessus and recapitulate the phenotypic effects of genetically disrupting dosS. OZ439 displayed bactericidal activity comparable to standard-of-care antibiotics in chronically infected mice, in addition to potentiating the activity of antibiotics used in combination. The identification of antimalarial drugs as potent inhibitors and adjunct inhibitors of M. abscessus in vivo offers repurposing opportunities that could have an immediate impact in the clinic.
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Antimaláricos , Infecciones por Mycobacterium no Tuberculosas , Mycobacterium abscessus , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antimaláricos/farmacología , Antimaláricos/uso terapéutico , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium abscessus/fisiologíaRESUMEN
The lengthy treatment time for tuberculosis (TB) is a primary cause for the emergence of multidrug resistant tuberculosis (MDR-TB). One approach to improve TB therapy is to develop an inhalational TB therapy that when administered in combination with oral TB drugs eases and shortens treatment. Spectinamides are new semisynthetic analogues of spectinomycin with excellent activity against Mycobacterium tuberculosis (Mtb), including MDR and XDR Mtb strains. Spectinamide-1599 was chosen as a promising candidate for development of inhalational therapy. Using the murine TB model and intrapulmonary aerosol delivery of spectinamide-1599, we characterized the pharmacokinetics and efficacy of this therapy in BALB/c and C3HeB/FeJ mice infected with the Mtb Erdman strain. As expected, spectinamide-1599 exhibited dose-dependent exposure in plasma, lungs, and ELF, but exposure ratios between lung and plasma were 12-40 times higher for intrapulmonary compared to intravenous or subcutaneous administration. In chronically infected BALB/c mice, low doses (10 mg/kg) of spectinamide-1599 when administered thrice weekly for two months provide efficacy similar to that of higher doses (50-100 mg/kg) after one month of therapy. In the C3HeB/FeJ TB model, intrapulmonary aerosol delivery of spectinamide-1599 (50 mg/kg) or oral pyrazinamide (150 mg/kg) had limited or no efficacy in monotherapy, but when both drugs were given in combination, a synergistic effect with superior bacterial reduction of >1.8 log10 CFU was observed. Throughout the up to eight-week treatment period, intrapulmonary therapy was well-tolerated without any overt toxicity. Overall, these results strongly support the further development of intrapulmonary spectinamide-1599 as a combination partner for anti-TB therapy.
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Espectinomicina , Tuberculosis , Animales , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Pirazinamida , Espectinomicina/farmacología , Tuberculosis/tratamiento farmacológicoRESUMEN
Pharmacokinetic/pharmacodynamic studies of anti-tuberculosis agents in animal models of tuberculosis are hampered by the frequent necessity to perform sample bioanalysis outside the biosafety level-3 environment. Thus, each specimen has to undergo tedious and time-consuming sample sterilization procedures that may also affect drug stability. Here, we tested treatment of Mycobacterium tuberculosis (Mtb) infected samples with methanol to sterilize samples while preserving drug integrity for further pharmacokinetic/pharmacodynamic evaluations. Tissue samples harvested from Mtb infected mice were homogenized, incubated in methanol, and tested for sterility. Once sterility was confirmed, the samples were used to determine concentrations of the anti-tuberculosis drug spectinamide-1599 in lung homogenates using liquid chromatography coupled with mass spectrometry. The results demonstrate that methanol sterilizes tissue samples harvested from Mtb infected mice without altering the integrity of the drug in the tissue.