RESUMO
BACKGROUND: Pulmonary tuberculosis is a chronic infectious disease of the respiratory system. It is still one of the leading causes of death from a single infectious disease, but it has been stuck in the study of a single pathogen. Recent studies have shown that many diseases are associated with disruption of the native microbiota. In this study we investigated the occurrence of tuberculosis and the correlation between drug resistance and respiratory flora. High-throughput 16 S rRNA gene sequencing was used to characterize the respiratory microbiota composition of 30 tuberculosis (TB) affected patients and compared with 30 healthy (H) controls. According to their Gene Xpert results, 30 pulmonary tuberculosis patients were divided into 12 persons in the drug-sensitive group (DS0) and 18 persons in the drug-resistant group (DR0). The microbial flora of the two were compared with the H group. RESULTS: The data generated by sequencing showed that Firmicutes, Proteus, Bacteroides, Actinomyces and Fusobacterium were the five main bacterial phyla detected, and they constituted more than 96% of the microbial community. The relative abundances of Fusobacterium, Haemophilus, Porphyromonas, Neisseria, TM7, Spirochetes, SR1, and Tenericutes in the TB group was lower than that of the H group, and Granulicatella was higher than the H group. The PcoA diagrams of the two groups had obvious clustering differences. The Alpha diversity of the TB group was lower than that of the H group, and the Beta diversity was higher than that of the H group (P < 0.05). The relative abundance of Streptococcus in the DS0 group was significantly higher than that in the DR0 group (P < 0.05). CONCLUSION: Pulmonary tuberculosis can cause disorders of the respiratory tract microbial flora, in which the relative abundance of Streptococcus was significantly different between rifampicin-sensitive and rifampicin-resistant patients.
Assuntos
Microbiota , Tuberculose Pulmonar , Humanos , Rifampina/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Sistema Respiratório , FusobacteriumRESUMO
Mycobacterium abscessus is intrinsically resistant to most antimicrobial agents. The emerging infections caused by M. abscessus and the lack of effective treatment call for rapid attention. Here, we intended to construct a selectable marker-free autoluminescent M. abscessus strain (designated UAlMab) as a real-time reporter strain to facilitate the discovery of effective drugs and regimens for treating M. abscessus The UAlMab strain was constructed using the dif/Xer recombinase system. In vitro and in vivo activities of several drugs, including clofazimine and TB47, a recently reported cytochrome bc1 inhibitor, were assessed using UAlMab. Furthermore, the efficacy of multiple drug combinations, including the clofazimine and TB47 combination, were tested against 20 clinical M. abscessus isolates. The UAlMab strain enabled us to evaluate drug efficacy both in vitro and in live BALB/c mice in a real-time, noninvasive fashion. Importantly, although TB47 showed marginal activity either alone or in combination with clarithromycin, amikacin, or roxithromycin, the drug markedly potentiated the activity of clofazimine, both in vitro and in vivo This study demonstrates that the use of the UAlMab strain can significantly facilitate rapid evaluation of new drugs and regimens. The clofazimine and TB47 combination is effective against M. abscessus, and dual/triple electron transport chain (ETC) targeting can be an effective therapeutic approach for treating mycobacterial infections.
Assuntos
Antibacterianos/farmacologia , Clofazimina/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Mycobacterium abscessus/efeitos dos fármacos , Amicacina/farmacologia , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Claritromicina/farmacologia , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Sinergismo Farmacológico , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Engenharia Genética/métodos , Luminescência , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/enzimologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/metabolismo , Imagem Óptica/métodos , Recombinases/genética , Recombinases/metabolismo , Roxitromicina/farmacologiaRESUMO
The sustained increase in the incidence of nontuberculous mycobacterial (NTM) infection and the difficulty in distinguishing these infections from tuberculosis constitute an urgent need for NTM species-level identification. The MeltPro Myco assay is the first diagnostic system that identifies 19 clinically relevant mycobacteria in a single reaction based on multicolor melting curve analysis run on a real-time PCR platform. The assay was comprehensively evaluated regarding its analytical and clinical performances. The MeltPro Myco assay accurately identified 51 reference mycobacterial strains to the species/genus level and showed no cross-reactivity with 16 nonmycobacterial strains. The limit of detection was 300 bacilli/ml, and 1% of the minor species was detected in the case of mixed infections. Clinical studies using 1,163 isolates collected from five geographically distinct health care units showed that the MeltPro Myco assay correctly identified 1,159 (99.7%) samples. Further testing with 94 smear-positive sputum samples showed that all samples were correctly identified. Additionally, the entire assay can be performed within 3 h. The results of this study confirmed the efficacy of this assay in the reliable identification of mycobacteria, suggesting that it might potentially be used as a screening tool in regions endemic for tuberculosis.
Assuntos
Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Micobactérias não Tuberculosas/isolamento & purificação , Coinfecção/diagnóstico , Coinfecção/microbiologia , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , Humanos , Técnicas Microbiológicas/normas , Técnicas de Diagnóstico Molecular/normas , Infecções por Mycobacterium não Tuberculosas/microbiologia , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Escarro/microbiologia , Fatores de Tempo , Tuberculose/diagnóstico , Tuberculose/microbiologiaRESUMO
BACKGROUND: Pulmonary nontuberculous mycobacteria (NTM) disease is of increasing public health concern in China. Information is limited regarding risk factors associated with this disease in China. The objective of this study was to describe the epidemiology of pulmonary disease due to NTM in Southern China. METHODS: We retrospectively reviewed the medical records of pulmonary NTM patients registered in the Guangzhou Chest Hospital with positive mycobacterial cultures during 2013-2016. We described sex, age, residence, treatment history, laboratory examination results and comorbidities of pulmonary NTM patients. RESULTS: Among the 607 NTM cases, the most prevalent species were Mycobacterium avium complex (44.5%), Mycobacterium abscessus complex (40.5%), Mycobacterium kansasii (10.0%) and Mycobacterium fortuitum (2.8%). The male:female ratio was significantly lower among patients infected with rapidly growing mycobacteria (RGM) than among those with slowly growing mycobacteria (SGM). The risk of developing SGM disease significantly increased with advancing age. In addition, pulmonary RGM diseases were more common in migrant population than resident population. Notably, patients with pulmonary RGM diseases were significantly more likely to have bronchiectasis underlying noted than those with SGM diseases. No significant difference was observed in in vitro drug susceptibility among NTM species. CONCLUSION: Our data illustrate that the M. avium complex is the most predominant causative agent of pulmonary NTM disease in Southern China. Female, migrant population, the presence of bronchiectasis are independent risk factors for pulmonary diseases due to RGM. In addition, the prevalence of SGM increases significantly with advancing age.
Assuntos
Pneumopatias/epidemiologia , Pneumopatias/microbiologia , Infecções por Mycobacterium não Tuberculosas/complicações , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Antibióticos Antituberculose/uso terapêutico , China/epidemiologia , Comorbidade , Feminino , Humanos , Pneumopatias/tratamento farmacológico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Micobactérias não Tuberculosas , Prevalência , Estudos Retrospectivos , Fatores de Risco , Distribuição por Sexo , Adulto JovemRESUMO
Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by Mycobacterium tuberculosis has been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers M. tuberculosis resistance to PZA. Expression of the mutant allele but not the wild-type allele in M. tuberculosis recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/análogos & derivados , Cromatografia Líquida de Alta Pressão , DNA de Cadeia Simples/genética , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Pirazinamida/farmacologia , Pirofosfatases/genéticaRESUMO
We expressed the wild-type rplC and mutated rplC (Cys154Arg) genes, respectively, in Mycobacterium tuberculosis H37Ra and H37Rv in an attempt to delineate the role of rplC (Cys154Arg) regarding oxazolidinone resistance. An increase of the MICs of linezolid (LZD) and sutezolid (PNU-100480, PNU) against the recombinant mycobacteria with overexpressed rplC mutation (Cys154Arg) was found, suggesting the rplC gene is a determinant of bacillary susceptibilities to LZD and PNU.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/metabolismo , Oxazolidinonas/farmacologia , Proteínas Ribossômicas/metabolismo , Linezolida/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Proteína Ribossômica L3 , Proteínas Ribossômicas/genéticaRESUMO
We sequenced pncA and rpsA genes plus flanking regions of 161 Mycobacterium tuberculosis isolates and found 10 new pncA and 3 novel rpsA mutations in pyrazinamide-resistant strains determined by the Bactec MGIT 960 system. The 3' end of rpsA might be added as the target of molecular detection of pyrazinamide susceptibility.
Assuntos
Amidoidrolases/genética , Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Pirazinamida/farmacologia , Proteínas Ribossômicas/genética , China , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/efeitos dos fármacos , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVE: To study regimens containing isoniazid and rifampicin for the treatment of pulmonary tuberculosis with isoniazid or rifampicin resistance. METHODS: Eighty patients with isoniazid or rifampicin resistance, and whose sputum were still positive at the end of 2-month therapy with isoniazid (H), rifampicin (R), pyrazineamide (Z) and ethambutol (E), were retrospectively analyzed from Jan.2009 to Dec.2012 in Guangzhou Chest Hospital. According to the Mycobacterium drug sensitive test (DST) before the treatment with isoniazid and rifampicin, the patients were divided into the sensitive group (either H or R sensitive), the multidrug-resistance group (both H and R resistance) and the single-resistance group (H or R resistance). There were 80 patients (57 females, 23 males) whose sputum was still positive at the end of 2 month treatment. Their ages ranged from 16-80 (average 45) years. Among them, 29 received the first-treatment, while 51 received retreatment. There were 37 cases in the sensitive group, with 18 first-treatment patients and 19 retreatment patients. There were 15 cases in the single-resistance group, with 3 first-treatment patients and 12 retreatment patients. There were 28 cases in the multidrug-resistance group, with 8 first-treatment patients and 20 retreatment patients. RESULTS: After treatment, mycobacterial conversion to MDR-TB occurred in 2 patients in the sensitive group, and in 6 patients in the single-resistance group. The rate of conversion to MDR-TB was higher in the single-resistance group than that in the sensitive group (χ² = 12.849, P = 0.000).Six patients with single H resistance converted to MDR-TB and 2 patients with single R resistance converted to MDR-TB (P < 0.05, RR = 18.0). CONCLUSIONS: Single H or R drug-resistance was more common in retreated patients with pulmonary tuberculosis.If regimens containing isoniazid and rifampicin was used to treat patients with single H or R drug-resistance, resistant enlarging effect may appear and lead to MDR-TB. The retreated patients should be monitored as soon as possible for detection of Mycobacterium resistance to H and R, and regimens for H or R-resistance should be used to prevent resistant enlarging effect.
Assuntos
Antituberculosos/uso terapêutico , Isoniazida/administração & dosagem , Rifampina/administração & dosagem , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Pulmonar/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Etambutol , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Retratamento , Estudos Retrospectivos , Escarro , Tórax , Resultado do Tratamento , Adulto JovemRESUMO
We aimed to study the distribution and contribution of mutations in the rpoB whole gene in rifampin-resistant/rifabutin-resistant (RIF(r)/Rfb(r)) (or RIF/Rfb cross-resistant) clinical Mycobacterium tuberculosis isolates. One standard M. tuberculosis strain (H37Rv) and 392 other clinical M. tuberculosis isolates mainly from Guangdong Province of China whose susceptibilities to rifampin (RIF), rifabutin (Rfb), streptomycin (SM), ethambutol (EMB), and isoniazid (INH) were previously determined were subjected to DNA sequencing of their rpoB whole genes. H37Rv and the 30 drug-susceptible clinical isolates had no mutations in rpoB whole genes. In 43 rifampin-resistant/rifabutin-susceptible (RIF(r)/Rfb(s)) isolates, the most frequent mutation codons were 516 (62.80%), 526 (14.0%), and 533 (6.98%), but codon 531 had no mutation. Twenty-one of the 43 isolates (48.84%) had single mutations of H526L, H526S, D516V, D516Y, and D516F. In 319 RIF(r)/Rfb(r) isolates, the most frequent mutation codons were 531 (73.7%) and 526 (18.8%); the mutation frequency for codon 516 was 2.5%, and that for codon 533 was only 0.31%. A total of 82.8% (264/319) of them had single mutations of S531L, S531W, H526D, H526Y, H526R, Q513K, Q513P, Q510H, V176F, P206(T)R, Y314(T)C, and H323(T)Y (the superscript T indicates M. tuberculosis numbering; the remaining codons use the E. coli numbering), among which V176F, P206(T)R, Y314(T)C, and H323(T)Y were located in the beginning of rpoB, and all of them were present in 1.9% (6/319) of RIF(r)/Rfb(r) isolates. The multiple mutations in RIF(r)/Rfb(r) isolates and in RIF(r)/Rfb(s) isolates were also different from each other either in mutation positions or in types of mutation combinations. In conclusion, the mutations of rpoB in RIF-R/Rfb(s) and in RIF-R/Rfb-R isolates differ significantly from each other not only in the most frequent mutation codons (516, 531, and 533) but also in the most frequent single mutations (S531L, H526L, D516V, D516Y, and D516F), and the beginning of rpoB may confer a RIF/Rfb cross-resistance phenotype in M. tuberculosis. Molecular assays for identifying RIF/Rfb cross-resistance in M. tuberculosis might be improved in terms of accuracy by including this region, in addition to the rifampin resistance determination region.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Mycobacterium tuberculosis/genética , Rifabutina/farmacologia , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antituberculosos/farmacologia , China , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Humanos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA , Adulto JovemRESUMO
OBJECTIVES: Amikacin is the only second-line injectable antituberculosis (anti-TB) drug still recommended for multidrug-resistant tuberculosis (MDR-TB) treatment when a short MDR-TB regimen is designed. Mutations in rrs and eis are reported to be associated with resistance to amikacin. In this study, we investigated the incidence of rrs, eis, tap and whiB7 mutations in amikacin-resistant Mycobacterium tuberculosis clinical isolates to find the proportion of different mutations related to amikacin resistance. METHODS: A total of 395 clinical isolates of M. tuberculosis were used for phenotypic drug susceptibility testing (DST) to 10 drugs with the Löwenstein-Jensen (L-J) method. We sequenced rrs, eis, tap and whiB7 genes in 178 M. tuberculosis clinical isolates (89 amikacin-resistant isolates and 89 of 306 amikacin-susceptible isolates). RESULTS: Our data showed that 22.53% (89/395) M. tuberculosis clinical isolates were resistant to amikacin. Of the 89 amikacin-resistant isolates, 89.89% (80/89) were MDR-TB, of which 12.36% (11/89) were pre-extensively drug-resistant TB (pre-XDR-TB) and 77.53% (69/89) were XDR-TB. The rrs mutations were found in 82% (73/89) in amikacin-resistant M. tuberculosis clinical isolates. The A1401G alteration in the rrs gene was the most dominant mutation (80.90%; 72/89). Five mutations were detected as new in rrs, tap and whiB7. Notably, 13.48% (12/89) amikacin-resistant isolates had no known mutation in these genes. CONCLUSIONS: Our data reveal that the rrs mutation is a predominant molecular marker of amikacin resistance in southern China. Analysis of the rrs gene mutations will significantly reduce the time and cost to diagnose amikacin resistance in TB patients. Other unknown amikacin resistance mechanism(s) exist.
Assuntos
Mycobacterium tuberculosis , Amicacina/farmacologia , Capreomicina , China/epidemiologia , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Canamicina , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , PrevalênciaRESUMO
Mycobacterium abscessus is a fast growing Mycobacterium species mainly causing skin and respiratory infections in human. M. abscessus is resistant to numerous drugs, which is a major challenge for the treatment. In this study, we have sequenced the genomes of two clinical M. abscessus strains having rough and smooth morphology, using the single molecule real-time and Illumina HiSeq sequencing technology. In addition, we reported the first comparative methylome profiles of a rough and a smooth M. abscessus clinical strains. The number of N4-methylcytosine (4mC) and N6-methyladenine (6mA) modified bases obtained from smooth phenotype were two-fold and 1.6 fold respectively higher than that of rough phenotype. We have also identified 4 distinct novel motifs in two clinical strains and genes encoding antibiotic-modifying/targeting enzymes and genes associated with intracellular survivability having different methylation patterns. To our knowledge, this is the first report about genome-wide methylation profiles of M. abscessus strains and identification of a natural linear plasmid (15 kb) in this critical pathogen harboring methylated bases. The pan-genome analysis of 25 M. abscessus strains including two clinical strains revealed an open pan genome comprises of 7596 gene clusters. Likewise, structural variation analysis revealed that the genome of rough phenotype strain contains more insertions and deletions than the smooth phenotype and that of the reference strain. A total of 391 single nucleotide variations responsible for the non-synonymous mutations were detected in clinical strains compared to the reference genome. The comparative genomic analysis elucidates the genome plasticity in this emerging pathogen. Furthermore, the detection of genome-wide methylation profiles of M. abscessus clinical strains may provide insight into the significant role of DNA methylation in pathogenicity and drug resistance in this opportunistic pathogen.
Assuntos
Epigenoma , Genoma Bacteriano , Mycobacterium abscessus/genética , Fenótipo , Metilação de DNA , Polimorfismo GenéticoRESUMO
Streptomycin (STR) is the first antibiotic used in the treatment of tuberculosis (TB) and the earliest antituberculosis drug with acquired resistance developed by Mycobacterium tuberculosis. The high prevalence of such resistance in many parts of the world limits its use for treating multidrug-resistant (MDR) TB. The aims of this study are to characterize of mutations in rpsL, rrs, and gidB genes in MDR M. tuberculosis isolates originating from southern China and to investigate possible relationship between mutations and strain genotypes for precise diagnosis and treatment. Sequences of rpsL, rrs, and gidB genes and the resistance profiles were analyzed for 218 MDR M. tuberculosis isolates. Our study showed that 68.35% of MDR M. tuberculosis isolates were resistant to STR and 89.91% of STR-resistant (STRR) isolates were Beijing lineage strains. Mutations were observed in STRR MDR M. tuberculosis isolates at the following rates: 72.48% in rpsL, 36.91% in rrs, and 15.44% in gidB. Compared with the phenotypic data, the combination of mutations in rpsL, rrs, and gidB has sensitivity and specificity of 96.64% and 100.00%, respectively. The most common mutations in STRR isolates were rpsL128,263 and rrs514,1401, of which rpsL128 showed association with Beijing lineage (p < 0.001). It is noteworthy that a1401g mutation was present in rrs, while MDR M. tuberculosis isolates were resistant to both STR and amikacin. Twenty two novel mutations were found in STRR isolates. These findings could be helpful to develop rapid molecular diagnostic methods and understand STR resistance in China for developing TB precision medicine and disturbance of drug-resistant TB transmission.
Assuntos
Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Estreptomicina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/genética , China/epidemiologia , Genes Bacterianos , Genótipo , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Toxin-antitoxin (TA) modules widely exist in bacteria, and their activities are associated with the persister phenotype of the pathogen Mycobacterium tuberculosis (M. tb). M. tb causes tuberculosis, a contagious and severe airborne disease. There are 10 MazEF TA systems in M. tb that play important roles in stress adaptation. How the antitoxins antagonize toxins in M. tb or how the 10 TA systems crosstalk to each other are of interest, but the detailed molecular mechanisms are largely unclear. MazEF-mt9 is a unique member among the MazEF family due to its tRNase activity, which is usually carried out by the VapC toxins. Here, we present the cocrystal structure of the MazEF-mt9 complex at 2.7 Å. By characterizing the association mode between the TA pairs through various techniques, we found that MazF-mt9 bound not only its cognate antitoxin but also the noncognate antitoxin MazE-mt1, a phenomenon that could be also observed in vivo. Based on our structural and biochemical work, we propose that the cognate and heterologous interactions among different TA systems work together in vivo to relieve the toxicity of MazF-mt9 toward M. tb cells.
Assuntos
Proteínas de Bactérias/química , Endorribonucleases/química , Mycobacterium tuberculosis/química , Estresse Fisiológico , Sistemas Toxina-Antitoxina , Adaptação Fisiológica , Proteínas de Bactérias/genética , Cristalização , Endorribonucleases/genética , Mycobacterium tuberculosis/genética , Ligação ProteicaRESUMO
Buruli ulcer (BU) is an emerging infectious disease that causes disfiguring skin ulcers. The causative agent, Mycobacterium ulcerans, secretes toxin called mycolactone that triggers inflammation and immunopathology. Existing treatments are lengthy and consist of drugs developed for tuberculosis. Here, we report that a pyrazolo[1,5-a]pyridine-3-carboxamide, TB47, is highly bactericidal against M. ulcerans both in vitro and in vivo. In the validated mouse model of BU, TB47 alone reduces M. ulcerans burden in mouse footpads by more than 2.5 log10 CFU compared to the standard BU treatment regimen recommended by the WHO. We show that mutations of ubiquinol-cytochrome C reductase cytochrome subunit B confer resistance to TB47 and the dissimilarity of CydABs from different mycobacteria may account for their differences in susceptibility to TB47. TB47 is highly potent against M. ulcerans and possesses desirable pharmacological attributes and low toxicity that warrant further assessment of this agent for treatment of BU.
Assuntos
Antibacterianos/uso terapêutico , Úlcera de Buruli/tratamento farmacológico , Úlcera de Buruli/microbiologia , Mycobacterium ulcerans/efeitos dos fármacos , Mycobacterium ulcerans/patogenicidade , Animais , Complexo III da Cadeia de Transporte de Elétrons/genética , Camundongos , Mutação , Mycobacterium ulcerans/genéticaRESUMO
OBJECTIVE: To explore the effects of systemic glucocorticoid treatment on tuberculous pleural effusion. METHODS: Ninety Wistar rats were intrapleurally injected with 0.03 mg of standard human Mycobacterium tuberculosis to establish models of tuberculous pleural effusions and then were randomly divided into 2 equal groups both without anti-tuberculosis treatment: glucocorticoids group (GG) undergoing intramuscular injection of 0.3 mg triamcinolone acetonide in the right thigh 24 h after intrapleural injection, and control group (CG) received nothing as control. 8, 24, 32, and 48 hours, and 3, 5, 7, 10, and 15 days after intramuscular injection 5 rats from each group were killed. The thorax was opened, the amount of pleural effusion (PE) was recorded, and the pleural cavity, histopathology of pleura and lung parenchyma were examined. The white blood cell (WBC) count and differential leukocyte count, and levels of total protein (TP), glucose (GLU), and lactic dehydrogenase (LDH) in the PE were determined. Bioassays were used to detect the PE levels of soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor beta1 (TGF-beta1), and interferon gamma (IFN-gamma). RESULTS: The PE volumes of GG 8 - 48 h after the intramuscular injection were significantly lower than those of CG (P < 0.05, P < 0.01), and PE completely disappeared on day 3. The WBC in PE 24 - 48 h after and the percentages of neutrophils 8 - 48 h after the intramuscular injection of GG were all significant lower than those of CG (all P < 0.01). The TP levels 32 and 48 h after the intramuscular injection of GG were both significantly higher than those of CG (both P < 0.01). The LDH level of GG within 24 h after the intramuscular injection was significantly lower than that of CG (P < 0.01). Both the sICAM-1 and TGF-beta1 levels of GG were higher 8 h after the intramuscular injection, but lower 48 h after the intramuscular injection than those of CG (both P < 0.01). The IFN-gamma levels 8 - 48 h after the intramuscular injection of GG were all higher than those of CC (all P < 0.01). The IFN-gamma/TGF-beta1 ratios at different time points of GG were all higher than those of CG, and there were significant differences in those 8 - 48 h after the intramuscular injection between these 2 groups (all P < 0.01). Pathologically, the mean thickness of pleura in GG was significantly less than that in CG. Congestion and edema in subpleural and pulmonary interstitium were less in GG than in CG. CONCLUSION: Early use of glucocorticoids helps reduce the inflammatory response in pleural cavity in tuberculous pleurisy accelerate the absorption of pleural effusion and decrease the thickness of pleura.
Assuntos
Glucocorticoides/uso terapêutico , Derrame Pleural/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Humanos , Derrame Pleural/etiologia , Distribuição Aleatória , Ratos , Ratos Wistar , Tuberculose Pleural/complicaçõesRESUMO
We have conducted a multicenter study of the diagnostic accuracy of the MTBDRplus 2.0 assay in compared with conventional and molecular reference standard in four tuberculosis (TB)-specialized hospitals of China. A total of 5038 patients were enrolled in this study. The overall sensitivity of the assay for the diagnosis of TB was 92.7% [1723/1858, 95% confidence interval (95% CI): 91.5-93.9]. In smear-positive/culture-positive cases the sensitivity was 97.7% (995/1018, 95% CI: 96.6-98.6), whereas in smear-negative/culture-positive cases it was 86.7% (728/840, 95% CI: 84.2-88.9). The agreement rate between MTBDRplus 2.0 and Xpert MTB/RIF was 97.7% (1015/1039, 95% CI: 96.6-98.5) for smear-positive cases and 97.0% (3682/3794, 95% CI: 96.5-97.6) for smear-negative cases. As compared with phenotypic drug susceptibility testing, the MTBDRplus 2.0 correctly identified 298 of 315 patients (94.6%, 95% CI: 91.5-96.8) with rifampicin-resistance. As noted previously, isoniazid resistance is associated with many different mutations and consequently the sensitivity compared to phenotypic testing was lower (81.0%, 95% CI: 76.8-84.7). In conclusion, this assay is a rapid, accurate test in terms of increased sensitivity for detecting smear-negative TB patients, as well as an alternative for detecting both RIF and INH resistance in persons with presumptive TB, whereas the absence of a mutation in the specimens must be interpreted cautiously.
Assuntos
Antituberculosos/uso terapêutico , DNA Bacteriano/análise , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Pulmonar/microbiologia , DNA Bacteriano/genética , Técnicas de Genotipagem , Humanos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológicoRESUMO
New diagnostic methods have provided a promising solution for rapid and reliable detection of drug-resistant TB strains. The aim of this study was to evaluate the performance of the MeltPro TB assay in identifying multidrug-resistant (MDR-) and extensively drug-resistant tuberculosis (XDR-TB) patients from sputum samples. The MeltPro TB assay was evaluated using sputum samples from 2057 smear-positive TB patients. Phenotypic Mycobacterial Growth Indicator Tube (MGIT) 960 drug susceptibility testing served as a reference standard. The sensitivity of the MeltPro TB assay was 94.2% for detecting resistance to rifampicin and 84.9% for detecting resistance to isoniazid. For second-line drugs, the assay showed a sensitivity of 83.3% for ofloxacin resistance, 75.0% for amikacin resistance, and 63.5% for kanamycin resistance. However, there was a significant difference for detecting kanamycin resistance between the two pilot sites in sensitivity, which was 53.2% in Guangdong and 81.5% in Shandong (P = 0.015). Overall, the MeltPro TB assay demonstrated good performance for the detection of MDR- and XDR-TB, with a sensitivity of 86.7% and 71.4%, respectively. The MeltPro TB assay is an excellent alternative for the detection of MDR- and XDR-TB cases in China, with high accuracy, short testing turn-around time, and low unit price compared with other tests.
Assuntos
Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Amicacina/farmacologia , China , Humanos , Canamicina/farmacologia , Ofloxacino/farmacologia , Rifampina/farmacologia , Sensibilidade e Especificidade , Escarro/microbiologiaRESUMO
OBJECTIVE: To evaluate the accuracy and applicability of species identification method of Mycobacteria by applying gas chromatography analysis of whole-cell fatty acid. METHODS: Species identification of 14 reference strains and 727 clinical isolates of Mycobacteria were performed by using MIDI Sherlock Microbial Identification System (MIS)4.0 based on gas chromatography analysis of whole-cell fatty acid, and the results were compared with those of conventional species identification method. RESULTS: (1) By using the conventional method, all 14 reference strains were identified correctly. Except for Mycobacterium.vaccae, the result obtained by MIS were identical to that of conventional method. (2) Among 625 clinical isolates identified as Mycobacterium tuberculosis (MTB) and Mycobacterium bovis, 45 strains were mistakenly identified as Nontuberculous Mycobacteria (NTM) by MIS. Its accuracy was 93%. For 102 NTM strains, results of group identification showed no difference between the two methods. However, 7 results of species identification were not consistent, the accuracy being 93%. (3) MIS mainly mistook MTB for Mycobacterium gastri, Mycobacterium trivial and Mycobacterium smegmatis, and Mycobacterium scrofulaceum for Mycobacterium gordonae among NTM. CONCLUSIONS: The results of species identification of Mycobacteria by applying gas chromatography analysis of whole-cell fatty acid are in accordance with those of conventional method for the majority of the strains, and MIS can identify Mycobacteria to species level by a single experiment. It can be considered as a good method for species identification of Mycobacteria in terms of its accuracy and applicability.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Cromatografia Gasosa/métodos , Ácidos Graxos/análise , Mycobacterium/isolamento & purificação , Humanos , Mycobacterium/classificação , Especificidade da EspécieRESUMO
OBJECTIVE: To develop a rat model of tuberculous pleurisy and to explore the mechanism of intrapleural inflammatory and immunological responses. METHODS: Fifty Wistar rats were injected intrapleurally with 0.03 mg of standard human mycobacterium tuberculous bacilli H37Rv each. The rats were killed in group on days 1, 2, 3, 5, 7, 10, 15, 20, 30 and 60 after the day of intrapleural injection. The thorax was opened and the amount of pleural effusion was recorded, and histopathology of pleural tissues and lung tissues were observed. The white blood cell (WBC) count and differentials, levels of total protein (TP), glucose (GLU) and lactic dehydrogenase (LDH) of pleural effusions were determined. Pleural fluid was analyzed for the levels of soluble intercellular adhesion molecule-1 (sICAM-1), transforming growth factor beta1 (TGF-beta1) and interferon gamma (IFN-gamma) by using appropriate bioassays. Ten rats were intrapleurally received 2 ml of normal saline and another 10 rats received 2 ml of undiluted PPD solution each as control. RESULTS: Bilateral pleural effusions appeared within 15 days in all rats intrapleurally received tuberculous bacilli. The peak amount of pleural fluid was on day 5 (6.7 +/- 0.5 ml). The neutrophils were the predominant cells for the first 24 hours, and then were followed by lymphocytes. In the pleural fluid, total protein concentration was between 51-55 g/L. The levels of glucose and LDH were 5.2 mmol/L and 18.1 micromol.s(-1).L(-1) on day 1 and changed to 2.8 mmol/L and 28.9 micromol.s(-1).L(-1) on day 15 respectively. The biochemistry parameters were in accordance with characteristics of tuberculous pleurisy. The sICAM-1 level increased early (21.9 ng/ml on day 1) and peaked on day 3 (38.0 ng/ml), then decreased over time (4.4 ng/ml on day 15). The level of IFN-gamma was 41.2 pg/ml on day 1 and increased and maintained at high levels over time. TGF-beta1 levels increased and peaked on day 7 (47.2 ng/ml), and then on day 15 decreased to a level lower than that of day 1. The ratio of IFN-gamma/TGF-beta1 increased from 1.32 on day 1 to 5.69 on day 15. Correlation analysis showed that sICAM-1 and IFN-gamma were closely related with WBC count and its differentials, as well as with LDH levels. Histopathological study revealed early pleural inflammation and late caseation. CONCLUSIONS: Wistar rats can be used as an experimental model for tuberculous pleurisy. Tuberculous inflammatory and immunological responses in acute tuberculous pleurisy is enhanced rather than suppressed.