Asunto(s)
Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Humanos , Mycobacterium tuberculosis/genética , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Mutación , Proteínas Bacterianas/genética , Pruebas de Sensibilidad Microbiana , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Quinolonas , Antituberculosos/farmacología , Citocromos/antagonistas & inhibidores , Complejo IV de Transporte de Electrones/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Quinolonas/farmacologíaRESUMEN
As acknowledged by the Clinical and Laboratory Standards Institute (CLSI), there is an insufficient evidence base on which to recommend a standard method for antimicrobial susceptibility testing against M. ulcerans. The agar proportion method has been recognized as the standard method for susceptibility testing against Mycobacterium tuberculosis complex (MTBC) isolates for decades (Woods GL, Engenack NL, Lin G, Turnidge JD (2018) CLSI standards: guidelines for health care excellence. Susceptibility testing of mycobacteria, Nocardia spp., and other aerobic Actinomycetes, 3rd edn. Clinical and Laboratory Standards Institute Copyright©2018 Clinical and Laboratory Standards Institute, Wayne (PA)). While it is more labor-intensive and requires larger amounts of drug or compound than broth-based testing, we recommend the agar proportion method for determination of minimum inhibitory concentrations against M. ulcerans. Herewith we present the method we implemented in our laboratory over the last 2 decades.
Asunto(s)
Mycobacterium ulcerans , Agar , Antibacterianos , Pruebas de Sensibilidad Microbiana , Preparaciones FarmacéuticasRESUMEN
Great progress has been made in understanding the pathogenesis and treatment of Buruli ulcer over the last 20 years. The rediscovery of the mouse footpad model of the disease with translation to clinical practice has changed treatment of this infectious disease, caused by Mycobacterium ulcerans, from surgery and skin grafting to the administration of antibiotics for 8 weeks or less with superior cure rates. Here we describe the development and enhancement of the mouse model during the last two decades.
Asunto(s)
Úlcera de Buruli , Mycobacterium ulcerans , Animales , Antibacterianos/farmacología , Úlcera de Buruli/tratamiento farmacológico , Modelos Animales de Enfermedad , Ratones , Preparaciones FarmacéuticasRESUMEN
Telacebec (Q203) is a new antituberculosis drug in clinical development that has extremely potent activity against Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU). The potency of Q203 has prompted investigation of its potential role in ultrashort, even single-dose, treatment regimens for BU in mouse models. However, the relationships of Q203 dose, dose schedule, duration, and host immune status to treatment outcomes remain unclear, as does the risk of emergence of drug resistance with Q203 monotherapy. Here, we used mouse footpad infection models in immunocompetent BALB/c and immunocompromised SCID-beige mice to compare different Q203 doses, different dosing schedules, and treatment durations ranging from 1 day to 2 weeks, on long-term outcomes. We also tested whether combining Q203 with a second drug can increase efficacy. Overall, efficacy depended on total dose more than on duration. Total doses of 5 to 20 mg/kg rendered nearly all BALB/c mice culture negative by 13 to 14 weeks posttreatment, without selection of Q203-resistant bacteria. Addition of a second drug did not significantly increase efficacy. Although less potent in SCID-beige mice, Q203 still rendered the majority of footpads culture negative at total doses of 10 to 20 mg/kg. Q203 resistance was identified in relapse isolates from some SCID-beige mice receiving monotherapy but not in isolates from those receiving Q203 combined with bedaquiline or clofazimine. Overall, these results support the potential of Q203 monotherapy for single-dose or other ultrashort therapy for BU, although highly immunocompromised hosts may require higher doses or durations and/or combination therapy.
Asunto(s)
Úlcera de Buruli , Mycobacterium ulcerans , Animales , Úlcera de Buruli/tratamiento farmacológico , Imidazoles , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Piperidinas , PiridinasRESUMEN
Telacebec (Q203) is a new antitubercular drug with extremely potent activity against Mycobacterium ulcerans Here, we explored the treatment-shortening potential of Q203 alone or in combination with rifampin (RIF) in a mouse footpad infection model. The first study compared Q203 at 5 and 10 mg/kg doses alone and with rifampin. Q203 alone rendered most mouse footpads culture negative in 2 weeks. Combining Q203 with rifampin resulted in a relapse-free cure 24 weeks after completing 2 weeks of treatment, compared to a 25% relapse rate in mice receiving RIF with clarithromycin, the current standard of care, for 4 weeks. The second study explored the dose-ranging activity of Q203 alone and with RIF, including the extended activity of Q203 after treatment discontinuation. The bactericidal activity of Q203 persisted for ≥ 4 weeks beyond the last dose. All mice receiving just 1 week of Q203 at 2 to 10 mg/kg were culture negative 4 weeks after stopping treatment. Mice receiving 2 weeks of Q203 at 0.5, 2, and 10 mg/kg were culture negative 4 weeks after treatment. RIF did not increase the efficacy of Q203. A pharmacokinetics substudy revealed that Q203 doses of 2 to 10 mg/kg in mice produce plasma concentrations similar to those produced by 100 to 300 mg doses in humans, with no adverse effect of RIF on Q203 concentrations. These results indicate the extraordinary potential of Q203 to reduce the duration of treatment necessary for a cure to ≤ 1 week (or 5 doses of 2 to 10 mg/kg) in our mouse footpad infection model and warrant further evaluation of Q203 in clinical trials.
Asunto(s)
Úlcera de Buruli , Mycobacterium ulcerans , Animales , Antibacterianos/uso terapéutico , Úlcera de Buruli/tratamiento farmacológico , Quimioterapia Combinada , Imidazoles , Ratones , Ratones Endogámicos BALB C , Piperidinas , PiridinasRESUMEN
Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among ~15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas , Farmacorresistencia Bacteriana , Mutación , Mycobacterium tuberculosis , Nitroimidazoles/farmacología , Nitrorreductasas , Oxazoles/farmacología , Ingeniería de Proteínas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Nitrorreductasas/genética , Nitrorreductasas/metabolismo , Polimorfismo GenéticoRESUMEN
Buruli ulcer is treatable with antibiotics. An 8-week course of rifampin (RIF) and either streptomycin (STR) or clarithromycin (CLR) cures over 90% of patients. However, STR requires injections and may be toxic, and CLR shares an adverse drug-drug interaction with RIF and may be poorly tolerated. Studies in a mouse footpad infection model showed that increasing the dose of RIF or using the long-acting rifamycin rifapentine (RPT), in combination with clofazimine (CFZ), a relatively well-tolerated antibiotic, can shorten treatment to 4 weeks. CFZ is reduced by a component of the electron transport chain (ETC) to produce reactive oxygen species toxic to bacteria. Synergistic activity of CFZ with other ETC-targeting drugs, the ATP synthase inhibitor bedaquiline (BDQ) and the bc1:aa3 oxidase inhibitor Q203 (now named telacebec), was recently described against Mycobacterium tuberculosis Recognizing that M. tuberculosis mutants lacking the alternative bd oxidase are hypersusceptible to Q203 and that Mycobacterium ulcerans is a natural bd oxidase-deficient mutant, we tested the in vitro susceptibility of M. ulcerans to Q203 and evaluated the treatment-shortening potential of novel 3- and 4-drug regimens combining RPT, CFZ, Q203, and/or BDQ in a mouse footpad model. The MIC of Q203 was extremely low (0.000075 to 0.00015 µg/ml). Footpad swelling decreased more rapidly in mice treated with Q203-containing regimens than in mice treated with RIF and STR (RIF+STR) and RPT and CFZ (RPT+CFZ). Nearly all footpads were culture negative after only 2 weeks of treatment with regimens containing RPT, CFZ, and Q203. No relapse was detected after only 2 weeks of treatment in mice treated with any of the Q203-containing regimens. In contrast, 15% of mice receiving RIF+STR for 4 weeks relapsed. We conclude that it may be possible to cure patients with Buruli ulcer in 14 days or less using Q203-containing regimens rather than currently recommended 56-day regimens.
Asunto(s)
Antibacterianos/farmacología , Úlcera de Buruli/tratamiento farmacológico , Mycobacterium ulcerans/efectos de los fármacos , Animales , Carga Bacteriana , Úlcera de Buruli/microbiología , Úlcera de Buruli/patología , Claritromicina/farmacología , Clofazimina/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana/efectos de los fármacos , Quimioterapia Combinada , Transporte de Electrón/efectos de los fármacos , Humanos , Imidazoles/farmacología , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium ulcerans/genética , Piperidinas/farmacología , Piridinas/farmacología , Rifampin/análogos & derivados , Rifampin/farmacología , Estreptomicina/farmacologíaRESUMEN
Novel regimens combining bedaquiline and pretomanid with either linezolid (BPaL regimen) or moxifloxacin and pyrazinamide (BPaMZ regimen) shorten the treatment duration needed to cure tuberculosis (TB) in BALB/c mice compared to that of the first-line regimen and have yielded promising results in initial clinical trials. However, the independent contribution of the investigational new drug pretomanid to the efficacy of BPaMZ has not been examined, and its contribution to BPaL has been examined only over the first 2 months of treatment. In the present study, the addition of pretomanid to BL increased bactericidal activity, prevented emergence of bedaquiline resistance, and shortened the duration needed to prevent relapse with drug-susceptible isolates by at least 2 months in BALB/c mice. Addition of pretomanid to bedaquiline, moxifloxacin, and pyrazinamide (BMZ) resulted in a 1-log10 greater CFU reduction after 1 month of treatment and/or reduced the number of mice relapsing in each of 2 experiments in BALB/c mice and in immunocompromised nude mice. Bedaquiline-resistant isolates were found at relapse in only one BMZ-treated nude mouse. Treatment of infection with a pyrazinamide-resistant mutant in BALB/c mice with BPaMZ prevented selection of bedaquiline-resistant mutants and reduced the proportion of mice relapsing compared to that for BMZ treatment alone. Among severely ill C3HeB/FeJ mice with caseous pneumonia and cavitation, BPaMZ increased median survival (≥60 versus 21 days) and reduced median lung CFU by 2.4 log10 at 1 month compared to the level for BMZ. In conclusion, in 3 different mouse models, pretomanid contributed significantly to the efficacy of the BPaMZ and BPaL regimens, including restricting the selection of bedaquiline-resistant mutants.
Asunto(s)
Antituberculosos/uso terapéutico , Diarilquinolinas/uso terapéutico , Linezolid/uso terapéutico , Moxifloxacino/uso terapéutico , Nitroimidazoles/uso terapéutico , Pirazinamida/uso terapéutico , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Ribosómico 16S/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/genéticaRESUMEN
Rifampin (RIF) plus clarithromycin (CLR) for 8 weeks is now the standard of care for Buruli ulcer (BU) treatment, but CLR may not be an ideal companion for rifamycins due to bidirectional drug-drug interactions. The oxazolidinone linezolid (LZD) was previously shown to be active against Mycobacterium ulcerans infection in mice but has dose- and duration-dependent toxicity in humans. Sutezolid (SZD) and tedizolid (TZD) may be safer than LZD. Here, we evaluated the efficacy of these oxazolidinones in combination with rifampin in a murine BU model. Mice with M. ulcerans-infected footpads received control regimens of RIF plus either streptomycin (STR) or CLR or test regimens of RIF plus either LZD (1 of 2 doses), SZD, or TZD for up to 8 weeks. All combination regimens reduced the swelling and bacterial burden in footpads after two weeks of treatment compared with RIF alone. RIF+SZD was the most active test regimen, while RIF+LZD was also no less active than RIF+CLR. After 4 and 6 weeks of treatment, neither CLR nor the oxazolidinones added significant bactericidal activity to RIF alone. By the end of 8 weeks of treatment, all regimens rendered footpads culture negative. We conclude that SZD and LZD warrant consideration as alternative companion agents to CLR in combination with RIF to treat BU, especially when CLR is contraindicated, intolerable, or unavailable. Further evaluation could prove SZD superior to CLR in this combination.
Asunto(s)
Antibacterianos/uso terapéutico , Úlcera de Buruli/tratamiento farmacológico , Mycobacterium ulcerans/efectos de los fármacos , Oxazolidinonas/uso terapéutico , Tetrazoles/uso terapéutico , Animales , Claritromicina/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Linezolid/efectos adversos , Linezolid/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Oxazolidinonas/efectos adversos , Rifampin/uso terapéutico , Tetrazoles/efectos adversosRESUMEN
TRIAL REGISTRATION: ClinicalTrials.gov NCT03474198, NCT01659437.
Asunto(s)
Úlcera de Buruli/tratamiento farmacológico , Clofazimina/uso terapéutico , Mycobacterium ulcerans/efectos de los fármacos , Rifamicinas/uso terapéutico , Animales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Clofazimina/administración & dosificación , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Quimioterapia Combinada , Ratones , Ratones Endogámicos BALB C , Rifamicinas/administración & dosificaciónRESUMEN
The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations.
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Antituberculosos/uso terapéutico , Clofazimina/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB C , Distribución Aleatoria , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
Drug efflux pumps play important roles in intrinsic and acquired drug resistance. Verapamil, an efflux inhibitor that enhances the activity of bedaquiline, clofazimine, and other drugs against Mycobacterium tuberculosis, has been proposed as a potential adjunctive agent for treatment of tuberculosis (TB). However, the extent to which verapamil enhances in vivo efficacy by inhibiting bacterial efflux pumps versus inhibiting mammalian drug transporters to improve oral bioavailability has not been delineated. We found that verapamil potentiated the in vitro activity of bedaquiline and clofazimine against M. tuberculosis clinical isolates, including those harboring rv0678 mutations. Verapamil increased the efficacy of bedaquiline in a murine TB model by the same extent to which it increased systemic bedaquiline exposure. However, verapamil showed no effect on the oral bioavailability or efficacy of clofazimine in mice. The addition of verapamil increased the sterilizing activity of a regimen composed of bedaquiline, clofazimine, and pyrazinamide. These results confirm that verapamil has adjunctive activity in vivo, but they also demonstrate that the adjunctive effect is likely due to enhanced systemic exposure to companion drugs via effects on mammalian transporters, rather than inhibition of bacterial pumps. Therefore, there may be no advantage to administering verapamil versus increasing the doses of companion drugs.
Asunto(s)
Antituberculosos/farmacocinética , Antituberculosos/uso terapéutico , Bloqueadores de los Canales de Calcio/farmacología , Clofazimina/farmacocinética , Clofazimina/uso terapéutico , Diarilquinolinas/farmacocinética , Diarilquinolinas/uso terapéutico , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiología , Verapamilo/farmacología , Animales , Disponibilidad Biológica , Recuento de Colonia Microbiana , Farmacorresistencia Bacteriana/efectos de los fármacos , Femenino , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacosRESUMEN
OBJECTIVES: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis. METHODS: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control. RESULTS: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered. CONCLUSIONS: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors.
Asunto(s)
Antituberculosos/uso terapéutico , Carga Bacteriana/efectos de los fármacos , Clofazimina/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antituberculosos/farmacocinética , Clofazimina/farmacocinética , Isoniazida/uso terapéutico , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Tuberculosis Pulmonar/microbiologíaRESUMEN
A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.
Asunto(s)
Antibióticos Antituberculosos/farmacología , Cicloserina/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Alanina-Deshidrogenasa/genética , Alanina-Deshidrogenasa/metabolismo , Alanina Racemasa/genética , Antituberculosos , Farmacorresistencia Bacteriana/genética , Técnicas de Inactivación de Genes , Genoma Bacteriano , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/enzimologíaRESUMEN
Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-µg/ml MIC for M. tuberculosis Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug.
Asunto(s)
Antituberculosos/uso terapéutico , Clofazimina/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Combinación de Medicamentos , Quimioterapia Combinada , Etambutol/uso terapéutico , Femenino , Isoniazida/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/patogenicidad , Pirazinamida/uso terapéutico , Rifampin/uso terapéutico , Privación de TratamientoRESUMEN
BACKGROUND: The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents. METHODS AND FINDINGS: We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937-1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974-1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988-1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe. CONCLUSIONS: In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.
Asunto(s)
Antituberculosos/farmacología , Tuberculosis Extensivamente Resistente a Drogas/genética , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Adulto , Brotes de Enfermedades , Tuberculosis Extensivamente Resistente a Drogas/tratamiento farmacológico , Tuberculosis Extensivamente Resistente a Drogas/epidemiología , Femenino , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Análisis de Secuencia de ADN , Sudáfrica/epidemiologíaRESUMEN
The discovery of Streptomyces-produced streptomycin founded the age of tuberculosis therapy. Despite the subsequent development of a curative regimen for this disease, tuberculosis remains a worldwide problem, and the emergence of multidrug-resistant Mycobacterium tuberculosis has prioritized the need for new drugs. Here we show that new optimized derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo, by inhibiting the DNA polymerase sliding clamp DnaN. We discovered that resistance to griselimycins, occurring at very low frequency, is associated with amplification of a chromosomal segment containing dnaN, as well as the ori site. Our results demonstrate that griselimycins have high translational potential for tuberculosis treatment, validate DnaN as an antimicrobial target, and capture the process of antibiotic pressure-induced gene amplification.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Terapia Molecular Dirigida , Mycobacterium tuberculosis/efectos de los fármacos , Péptidos Cíclicos/farmacología , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Animales , Antituberculosos/química , Antituberculosos/uso terapéutico , Línea Celular Tumoral , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN , Modelos Animales de Enfermedad , Diseño de Fármacos , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/enzimología , Péptidos Cíclicos/química , Péptidos Cíclicos/uso terapéutico , Estructura Secundaria de Proteína , Streptomyces/química , Streptomyces/efectos de los fármacos , Streptomyces/metabolismo , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
The antileprosy drug clofazimine has shown potential for shortening tuberculosis treatment; however, the current dosing of the drug is not evidence based, and the optimal dosing is unknown. Our objective was to conduct a preclinical evaluation of the pharmacokinetics and pharmacodynamics of clofazimine in the mouse model of tuberculosis, with the goal of providing useful information on dosing for future studies. Pharmacokinetic parameters were evaluated in infected and uninfected BALB/c mice. Pharmacodynamic parameters were evaluated in Mycobacterium tuberculosis-infected mice that were treated for 12 weeks with one of six different clofazimine dosing regimens, i.e., doses of 6.25, 12.5, and 25 mg/kg of body weight/day and 3 regimens with loading doses. Clofazimine progressively accumulated in the lungs, livers, and spleens of the mice, reaching levels of greater than 50 µg/g in all tissues by 4 weeks of administration, while serum drug levels remained low at 1 to 2 µg/ml. Elimination of clofazimine was extremely slow, and the half-life was dependent on the duration of drug administration. Clofazimine exhibited dose-dependent tissue and serum concentrations. At any dose, clofazimine did not have bactericidal activity during the first 2 weeks of administration but subsequently demonstrated potent, dose-independent bactericidal activity. The antituberculosis activity of clofazimine was dependent on neither the dose administered nor the drug concentrations in the tissues, suggesting that much lower doses could be effectively used for tuberculosis treatment.
Asunto(s)
Antituberculosos/farmacocinética , Clofazimina/farmacocinética , Tuberculosis/sangre , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/uso terapéutico , Cromatografía Liquida , Clofazimina/uso terapéutico , Femenino , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Unión ProteicaRESUMEN
The Mycobacterium avium complex is the most common cause of nontuberculous mycobacterial lung disease worldwide; yet, an optimal treatment regimen for M. avium complex infection has not been established. Clarithromycin is accepted as the cornerstone drug for treatment of M. avium lung disease; however, good model systems, especially animal models, are needed to evaluate the most effective companion drugs. We performed a series of experiments to evaluate and use different mouse models (comparing BALB/c, C57BL/6, nude, and beige mice) of M. avium infection and to assess the anti-M. avium activity of single and combination drug regimens, in vitro, ex vivo, and in mice. In vitro, clarithromycin and moxifloxacin were most active against M. avium, and no antagonism was observed between these two drugs. Nude mice were more susceptible to M. avium infection than the other mouse strains tested, but the impact of treatment was most clearly seen in M. avium-infected BALB/c mice. The combination of clarithromycin-ethambutol-rifampin was more effective in all infected mice than moxifloxacin-ethambutol-rifampin; the addition of moxifloxacin to the clarithromycin-containing regimen did not increase treatment efficacy. Clarithromycin-containing regimens are the most effective for M. avium infection; substitution of moxifloxacin for clarithromycin had a negative impact on treatment efficacy.