RESUMO
The activities of rifampin, rifapentine, bedaquiline, PA-824, clofazimine, nitazoxanide, isoniazid, amikacin, moxifloxacin, niclosamide, thioridazine, and pyrazinamide were tested against nonreplicating (dormant) Mycobacterium tuberculosis H37Rv under conditions of hypoxia at pHs 5.8 and 7.3, mimicking environments of cellular granulomas and caseous granulomas, respectively. At pH 5.8, several drugs killed dormant bacilli, with the best being rifampin and rifapentine. At pH 7.3, only rifampin and rifapentine efficiently killed dormant bacilli, while all other drugs showed little activity.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/análogos & derivados , Rifampina/farmacologia , Anaerobiose , Concentração de Íons de Hidrogênio , Ácidos Isonicotínicos/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Nitroimidazóis/farmacologia , Fenazinas/farmacologia , Quinolinas/farmacologia , Tiazóis/farmacologiaRESUMO
The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Radical Hidroxila/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Superóxidos/metabolismo , Antituberculosos/metabolismo , Proteínas de Bactérias/química , Catalase/química , Óxidos N-Cíclicos , RNA Polimerases Dirigidas por DNA , Espectroscopia de Ressonância de Spin Eletrônica , Radical Hidroxila/química , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Estresse Oxidativo , Ácido Pentético/química , Ligação Proteica , Rifampina/metabolismo , Marcadores de Spin , Superóxido Dismutase/química , Superóxidos/química , Tioureia/químicaRESUMO
Despite available global efforts and funding, Tuberculosis (TB) continues to affect a considerable number of patients worldwide. Policy makers and stakeholders set clear goals to reduce TB incidence and mortality, but the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) complicate the reach of these goals. Drug-resistance TB needs to be diagnosed rapidly and accurately to effectively treat patients, prevent the transmission of MDR-TB, minimise mortality, reduce treatment costs and avoid unnecessary hospitalisations. In this narrative review, we provide a comprehensive overview of laboratory methods for detecting drug resistance in MTB, focusing on phenotypic, molecular and other drug susceptibility testing (DST) techniques. We found a large variety of methods used, with the BACTEC MGIT 960 being the most common phenotypic DST and the Xpert MTB/RIF being the most common molecular DST. We emphasise the importance of integrating phenotypic and molecular DST to address issues like resistance to new drugs, heteroresistance, mixed infections and low-level resistance mutations. Notably, most of the analysed studies adhered to the outdated definition of XDR-TB and did not consider the pre-XDR definition, thus posing challenges in aligning diagnostic methods with the current landscape of TB resistance.
RESUMO
Mycobacterium tuberculosis is exposed to hypoxia and acidity within granulomatous lesions. In this study, an acidic culture model of M. tuberculosis was used to test drug activity against aerobic 5-day-old (A5) and hypoxic 5-, 12-, and 19-day-old (H5, H12, and H19, respectively) bacilli after 7, 14, and 21 days of exposure. In A cultures, CFU and pH rapidly increased, while in H cultures growth stopped and pH increased slightly. Ten drugs were tested: rifampin (R), isoniazid (I), pyrazinamide (Z), ethambutol (E), moxifloxacin (MX), amikacin (AK), metronidazole (MZ), nitazoxanide (NZ), niclosamide (NC), and PA-824 (PA). Rifampin was the most active against A5, H5, H12, and H19 bacilli. Moxifloxacin and AK efficiently killed A5 and H5 cells, I was active mostly against A5 cells, Z was most active against H12 and H19 cells, and E showed low activity. Among nitrocompounds, NZ, NC, and PA were effective against A5, H5, H12, and H19 cells, while MZ was active against H12 and H19 cells. To kill all A and H cells, A5- and H5-active agents R, MX, and AK were used in combination with MZ, NZ, NC, or PA, in comparison with R-I-Z-E, currently used for human therapy. Mycobacterial viability was determined by CFU and a sensitive test in broth (day to positivity, MGIT 960 system). As shown by lack of regrowth in MGIT, the most potent combination was R-MX-AK-PA, which killed all A5, H5, H12, and H19 cells in 14 days. These observations demonstrate the sterilizing effect of drug combinations against cells of different M. tuberculosis stages grown in aerobic and hypoxic acidic conditions.
Assuntos
Aerobiose/efeitos dos fármacos , Anaerobiose/efeitos dos fármacos , Antituberculosos/farmacologia , Combinação de Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Aerobiose/fisiologia , Anaerobiose/fisiologia , Contagem de Colônia Microbiana , Meios de Cultura , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimentoAssuntos
Emigrantes e Imigrantes/estatística & dados numéricos , Tuberculose Extensivamente Resistente a Medicamentos/epidemiologia , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Antituberculosos/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Humanos , Itália/epidemiologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
The susceptibility of 211 clinical isolates of Mycobacterium tuberculosis complex (201 M. tuberculosis and 10 Mycobacterium bovis isolates) to pyrazinamide (PZA) was assessed by the nonradiometric Bactec MGIT 960 system (M960). Detection of PZA resistance was followed by a repeat testing using a reduced inoculum (RI) of 0.25 ml instead of 0.5 ml. According to the first M960 analysis, resistance was observed in 55 samples. In the RI assay, 32 samples turned out to be susceptible and 23 proved to be resistant (58.2% false positivity). The Bactec 460 assay confirmed as resistant those strains detected by the RI assay, while discrepant results were found susceptible. Mutation analysis performed on 13 M. tuberculosis isolates detected pncA mutations in 11 samples. On the basis of our data, we suggest using the RI assay to confirm all PZA resistance results obtained with the standard M960 assay. Further studies are required to confirm our findings.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Reações Falso-Positivas , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/microbiologiaAssuntos
Antituberculosos/farmacologia , Erros de Diagnóstico/prevenção & controle , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Amidoidrolases/genética , Farmacorresistência Bacteriana/genética , Humanos , Mutação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/genética , Tuberculose/diagnóstico , Tuberculose/microbiologiaAssuntos
Migrantes , Tuberculose/epidemiologia , África , Antituberculosos/uso terapêutico , Europa (Continente) , Tuberculose Extensivamente Resistente a Medicamentos/epidemiologia , Geografia , Humanos , Itália/epidemiologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis , Federação Russa , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Organização Mundial da SaúdeRESUMO
Pyrazinamide (PZA) is a first-line key drug used in combination with other agents for the treatment of tuberculosis (TB). Phenotypic and molecular assays for testing susceptibility of Mycobacterium tuberculosis (Mtb) to PZA have been developed, with the assay in liquid medium at acidic pH in the Bactec MGIT 960 (M960) system being routinely used in the mycobacteriology laboratories. However, false resistance to PZA by this method was reported to occur by several investigators, mostly due to high Mtb inoculum, which may impair drug activity by increasing the pH of the medium. In this study, a revision of the literature on the issue of false resistance in the M960 PZA assay was performed. In the reports examined, all improvements of the M960 test proposed to decrease false resistant results were based on the use of reduced inoculum densities of Mtb cells, to be easily translated into laboratory practice.
Assuntos
Antituberculosos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Meios de Cultura/química , Farmacorresistência Bacteriana , Reações Falso-Positivas , Humanos , Concentração de Íons de Hidrogênio , Infecções por Mycobacterium/microbiologia , Reprodutibilidade dos Testes , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaAssuntos
Emigrantes e Imigrantes/estatística & dados numéricos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/etnologia , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adulto , Idoso , Controle de Doenças Transmissíveis , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Prevalência , Características de ResidênciaRESUMO
Current tuberculosis (TB) treatment requires 6 months of combination therapy with isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol for active TB and 9 months of INH or 3 months of rifapentine (RFP) + INH for latent TB. The lungs of patients with active and latent TB contain heterogeneous mixtures of cellular and caseous granulomas harboring Mycobacterium tuberculosis bacilli ranging from actively replicating (AR) to nonreplicating (NR), phenotypically drug-resistant stages. Several in vitro models to obtain NR cells were reported, including exposure to hypoxia, nutrient starvation, acid + nitric oxide, and stationary phase. Overall, these models showed that RIF, RFP, PA-824 (PA), metronidazole (MZ), bedaquiline (BQ), and fluoroquinolones were the most active drugs against NR M. tuberculosis. In hypoxia at pH 5.8, some combinations killed AR plus NR cells, as shown by lack of regrowth in liquid media, whereas in hypoxia at pH 7.3 (the pH of the caseum), only RIF and RFP efficiently killed NR bacilli while several other drugs showed little effect. In conventional mouse models, combinations containing RFP, BQ, PA, PZA, moxifloxacin, sutezolid, linezolid, and clofazimine sterilized animals in ≤2 months, as shown by lack of viable bacilli in lung homogenates after 3 months without therapy. Drugs were less effective in C3HeB/FeJ mice forming caseous granulomas. Overall, in vitro observations and in vivo studies suggest that the search for new TB drugs could be addressed to low lipophilic molecules (e.g., new rpoB inhibitors with clogP < 3) killing NR M. tuberculosis in hypoxia at neutral pH and reaching high rates of unbound drug in the caseum.
Assuntos
Antituberculosos/uso terapêutico , Tuberculose Latente/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia , Animais , Antituberculosos/farmacologia , Contagem de Colônia Microbiana , Humanos , Isoniazida/uso terapêutico , Tuberculose Latente/microbiologia , Camundongos , Mycobacterium tuberculosis/fisiologia , Pirazinamida/uso terapêutico , Rifampina/análogos & derivados , Rifampina/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.
Assuntos
Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Oxigênio/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Mycobacterium tuberculosis/metabolismo , RegulonRESUMO
BACKGROUND: Drug-resistant tuberculosis (TB) is a serious threat in industrialized countries, but information from Southern Italy is lacking. Here, we present the results of a retrospective study of TB cases diagnosed in 2008-2013 in Naples, the largest city in Southern Italy. METHODS: Six hundred ninety Mycobacterium tuberculosis strains were isolated at the Ospedali dei Colli of Naples, and resistance to first-line and second-line drugs was determined. RESULTS: Multidrug-resistant (MDR) TB increased from 2008 to 2013, with 77.4% of strains isolated from migrants from 41 countries. Overall, 4.5% of strains were MDR: Italian-born persons, 2.2%; Romania, 7.5%; Former Soviet Union countries (Ukraine, Russia, Armenia, Georgia), 22.4%; all other foreign countries, 2.0%. Resistance of MDR strains to second-line drugs was high against kanamycin, ofloxacin, capreomycin. CONCLUSIONS: MDR-TB in Naples increased in 2008-13 and was observed predominantly in migrants, indicating the need to intensify diagnosis and treatment of these populations in this town.
Assuntos
Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Criança , Pré-Escolar , Farmacorresistência Bacteriana Múltipla , Feminino , Humanos , Lactente , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/efeitos dos fármacos , Nepal/epidemiologia , Estudos Retrospectivos , Migrantes , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adulto JovemRESUMO
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), which kills about 2 million people annually. Furthermore, 2 billion people worldwide are latently infected with this organism, with 10% of them reactivating to active TB due to re-growth of nonreplicating (dormant) Mtb residing in their tissues. Because of the huge reservoir of latent TB it is important to find novel drugs/drug combinations killing dormant bacilli (microaerophiles, anaerobes and drug-tolerant persisters) surviving for decades in a wide spectrum of granulomatous lesions in the lungs of TB patients. Antibiotic treatment of drug-susceptible TB requires administration of isoniazid, rifampin, pyrazinamide, ethambutol for 2 months, followed by isoniazid and rifampin for 4 months. To avoid reactivation of dormant Mtb to active pulmonary TB, up to 9 months of treatment with isoniazid is required. Therefore, a strategy to eliminate dormant bacilli needs to be developed to shorten therapy of active and latent TB and reduce the reservoir of people with latent TB. Finding drugs with high rate of penetration into the caseous granulomas and understanding the biology of dormant bacilli and in particular of persister cells, phenotypically resistant to antibiotics, will be essential to eradicate Mtb from humans. In recent years unprecedented efforts have been done in TB drug discovery, aimed at identifying novel drugs and drug combinations killing both actively replicating and nonreplicating Mtb in vitro, in animal models and in clinical trials in humans.