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1.
PLoS One ; 15(8): e0238298, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32857802

RESUMO

BACKGROUND: The spread of multi-drug resistant tuberculosis (MDR-TB) is a leading global public-health challenge. Because not all biological mechanisms of resistance are known, culture-based (phenotypic) drug-susceptibility testing (DST) provides important information that influences clinical decision-making. Current phenotypic tests typically require pre-culture to ensure bacterial loads are at a testable level (taking 2-4 weeks) followed by 10-14 days to confirm growth or lack thereof. METHODS AND FINDINGS: We present a 2-step method to obtain DST results within 3 days of sample collection. The first involves selectively concentrating live mycobacterial cells present in relatively large volumes of sputum (~2-10mL) using commercially available magnetic-nanoparticles (MNPs) into smaller volumes, thereby bypassing the need for pre-culture. The second involves using microchannel Electrical Impedance Spectroscopy (m-EIS) to monitor multiple aliquots of small volumes (~10µL) of suspension containing mycobacterial cells, MNPs, and candidate-drugs to determine whether cells grow, die, or remain static under the conditions tested. m-EIS yields an estimate for the solution "bulk capacitance" (Cb), a parameter that is proportional to the number of live bacteria in suspension. We are thus able to detect cell death (bactericidal action of the drug) in addition to cell-growth. We demonstrate proof-of-principle using M. bovis BCG and M. smegmatis suspended in artificial sputum. Loads of ~ 2000-10,000 CFU of mycobacteria were extracted from ~5mL of artificial sputum during the decontamination process with efficiencies of 84% -100%. Subsequently, suspensions containing ~105 CFU/mL of mycobacteria with 10 mg/mL of MNPs were monitored in the presence of bacteriostatic and bactericidal drugs at concentrations below, at, and above known MIC (Minimum Inhibitory Concentration) values. m-EIS data (ΔCb) showed data consistent with growth, death or stasis as expected and/or recorded using plate counts. Electrical signals of death were visible as early as 3 hours, and growth was seen in < 3 days for all samples, allowing us to perform DST in < 3 days. CONCLUSION: We demonstrated "proof of principle" that (a) live mycobacteria can be isolated from sputum using MNPs with high efficiency (almost all the bacteria that survive decontamination) and (b) that the efficacy of candidate drugs on the mycobacteria thus isolated (in suspensions containing MNPs) could be tested in real-time using m-EIS.


Assuntos
Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana/métodos , Mycobacterium/efeitos dos fármacos , Escarro/microbiologia , Espectroscopia Dielétrica , Impedância Elétrica , Nanopartículas de Magnetita , Testes de Sensibilidade Microbiana/instrumentação , Mycobacterium/isolamento & purificação , Estudo de Prova de Conceito
2.
Proc Natl Acad Sci U S A ; 117(28): 16324-16332, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601219

RESUMO

FadE, an acyl-CoA dehydrogenase, introduces unsaturation to carbon chains in lipid metabolism pathways. Here, we report that FadE5 from Mycobacterium tuberculosis (MtbFadE5) and Mycobacterium smegmatis (MsFadE5) play roles in drug resistance and exhibit broad specificity for linear acyl-CoA substrates but have a preference for those with long carbon chains. Here, the structures of MsFadE5 and MtbFadE5, in the presence and absence of substrates, have been determined. These reveal the molecular basis for the broad substrate specificity of these enzymes. FadE5 interacts with the CoA region of the substrate through a large number of hydrogen bonds and an unusual π-π stacking interaction, allowing these enzymes to accept both short- and long-chain substrates. Residues in the substrate binding cavity reorient their side chains to accommodate substrates of various lengths. Longer carbon-chain substrates make more numerous hydrophobic interactions with the enzyme compared with the shorter-chain substrates, resulting in a preference for this type of substrate.


Assuntos
Acil-CoA Desidrogenase/química , Acil-CoA Desidrogenase/metabolismo , Mycobacterium/enzimologia , Acil Coenzima A/metabolismo , Acil-CoA Desidrogenase/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Domínio Catalítico , Farmacorresistência Bacteriana/genética , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Modelos Moleculares , Mutação , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
3.
Medicine (Baltimore) ; 99(16): e19697, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32311949

RESUMO

This study aims to elucidate the strains and drug resistance of mycobacterium isolated from osteoarticular tuberculosis (OATB) patients and provide a reference for the diagnosis and treatment of OATB.Sixty-nine clinically diagnosed and surgically treated OATB patients were collected in time period of January 2017 to December 2018 at the First Affiliated Hospital of Xinjiang Medical University. The BACTEC MGIT 960 system was used for mycobacteria culturing, strain identification, and drug susceptibility testing, and the mycobacteria culture positive rate, species distribution, and drug resistance were analyzed.Within 4 weeks, 24 (34.78%) isolates of mycobacteria culture were positive; 40 (57.97%) isolates were positive, when culturing time was expanded to 8 weeks, and the difference was statistically significant (P < .05). Among the 40 isolates, 24 (60%) were identified as mycobacterium tuberculosis (MTB), 10 (25%) were Mycobacterium bovis, and 6 (15%) were non-tuberculous mycobacteria (NTM). Among total 69 isolates, 40 were enrolled in drug sensitivity test, and 15 (37.5%) isolates were confirmed drug resistant strains, in which 5 isolates were MTB, 4 isolates were M. bovis, and 6 isolates of NTM.The pathogen of clinically diagnosed OATB was mainly MTB. However, M. bovis and NTM also accounted for a considerable proportion, and their drug resistance rate was higher. Extending the culturing time appropriately could improve the culture positive rate. NTM was a drug resistant strain, and mycobacteria culturing, strain identification, and drug resistance analysis should be carried out to serve as a guide for individual treatment.


Assuntos
Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana , Mycobacterium/efeitos dos fármacos , Tuberculose Osteoarticular/tratamento farmacológico , Tuberculose Osteoarticular/microbiologia , Adolescente , Adulto , Idoso , Criança , China , Feminino , Humanos , Masculino , Técnicas Microbiológicas , Pessoa de Meia-Idade , Mycobacterium/isolamento & purificação , Adulto Jovem
4.
Am J Case Rep ; 21: e921517, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32255770

RESUMO

BACKGROUND Nontuberculous mycobacteria (NTM) are environmental pathogens that cause an increasing number of diseases, in particular in immunosuppressed patients. Diagnosing NTM infections may be difficult because clinical presentation is unspecific and resembles other conditions such as tuberculosis, lymphomas, or septicemia. CASE REPORT We report the case of a 62-year-old male with a recent history of autologous bone marrow transplantation for a follicular lymphoma admitted to our department for long-lasting remittent fever and abscess-like splenic nodules. The patient was diagnosed with mixed systemic infection by Mycobacterium abscessus and Mycobacterium celatum localized in spleen, bone marrow and kidneys. CONCLUSIONS In this case a rare disseminated atypical mycobacteriosis was diagnosed and treated. As far as we know this is the first case in the literature of M. abscessus localization either in the spleen or in the bone marrow. Our patient underwent a complex long-term therapy and had a complete resolution of the disease.


Assuntos
Antibacterianos/uso terapêutico , Hospedeiro Imunocomprometido , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium/efeitos dos fármacos , Transplante de Medula Óssea , Humanos , Linfoma Folicular/imunologia , Masculino , Pessoa de Meia-Idade
5.
Int J Nanomedicine ; 15: 1073-1094, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32103956

RESUMO

Purpose: This study demonstrated improved transdermal delivery of rifampicin-loaded cationic nanoemulsion gel to treat systemic and cutaneous tuberculosis using capmul, labrasol, and acconon, which exert anti-Mycobacterium activities. This approach enhanced drug permeation across the skin, increased therapeutic efficacy, and reduced dose-related side effects. Methods: Design Expert® was used to optimize formulations (Smix ratio and capmul as independent factors), which were prepared using a slow spontaneous titration method. The optimized nanoemulsion was incorporated into carbopol gel to allow for topical application and comparative assessments. Nanoemulsions and gels were evaluated for size, size distribution, shape, zeta potential, percent spread, viscosity, in vitro hemolysis, in vitro release, and ex vivo skin permeation and deposition. A mechanistic evaluation was performed using scanning electron microscopy. Furthermore, in vivo pharmacokinetic and irritation studies were performed. Results: The optimized cationic nanoemulsion (OCNE-1) was characterized by small particle size (≤100 nm), had optimal viscosity, percent spread, zeta potential, and percent drug release, and was hemocompatible. The OCNE-1T gel exhibited higher permeation flux (51.32 ± 0.5 µg/cm2 hr), permeation coefficient (2.566 ± 0.08 cm/hr), drug deposition (994.404 µg/cm2), and enhancement ratio (7.16) than those of the OCNE-1 nanoemulsion or drug solution. Scanning electron microscopy was used to characterize the mechanism of enhanced permeation. An In vivo study showed that the Cmax and area under the curve following transdermal application were 4.34- and 4.74-fold higher than those following oral administration. Conclusion: Transdermal delivery of rifampicin could be a promising alternative to conventional approaches to treat systemic and local tuberculosis, and other bacterial infections.


Assuntos
Antituberculosos/administração & dosagem , Emulsões/administração & dosagem , Rifampina/administração & dosagem , Tuberculose/tratamento farmacológico , Resinas Acrílicas/química , Administração Cutânea , Animais , Antituberculosos/farmacocinética , Antituberculosos/farmacologia , Disponibilidade Biológica , Caprilatos/química , Cátions/química , Emulsões/farmacologia , Excipientes/química , Géis/química , Géis/farmacologia , Glicerídeos/química , Mycobacterium/efeitos dos fármacos , Nanoestruturas/administração & dosagem , Nanoestruturas/química , Tamanho da Partícula , Ratos Sprague-Dawley , Rifampina/farmacocinética , Rifampina/farmacologia , Pele/efeitos dos fármacos , Tuberculose Cutânea/tratamento farmacológico
6.
Eur J Med Chem ; 186: 111882, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31753514

RESUMO

A series of halogenated (4-methoxyphenyl)-1H-tetrazol-5-amine regioisomers (1a-9a, 1b-9b) were synthesized from their corresponding thiourea analogues (1-9). The synthesis pathway was confirmed by an X-ray crystallographic studies of 1a, 1b and 5a. Title derivatives were tested for their in vitro antitubercular activity against standard, "wild-type" and atypical mycobacteria. The highest therapeutic potential was attributed to isomeric N-(bromophenyl)tetrazoles 8a and 9a. Their growth-inhibitory effect against multidrug-resistant Mycobacterium tuberculosis Spec. 210 was 8-16-fold stronger than that of the first-line tuberculostatics. Other new tetrazole-derived compounds were also more or equally effective towards that pathogen comparing to the established pharmaceuticals. Among non-tuberculous strains, Mycobacterium scrofulaceum was the most susceptible to the presence of the majority of tetrazole derivatives. The synergistic interaction was found between 9a and streptomycin, as well as the additivity of both 8a and 9a in pairs with isoniazid, rifampicin and ethambutol. None of the studied compounds displayed antibacterial or cytotoxic properties against normal and cancer cell lines, which indicated their highly selective antimycobacterial effects.


Assuntos
Aminas/farmacologia , Antituberculosos/farmacologia , Mycobacterium/efeitos dos fármacos , Tetrazóis/farmacologia , Aminas/síntese química , Aminas/química , Antituberculosos/síntese química , Antituberculosos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/química
7.
J Antibiot (Tokyo) ; 73(1): 40-47, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31481764

RESUMO

Methylobacterium sp. is isolated from water distribution systems and has been linked in the biofilms of the systems with a lower presence of Mycobacterium avium. In this study we aimed to determine the in vitro activity of Methylobacterium sp. in the development of rapidly growing mycobacteria (RGM) biofilms. Methylobacterium sp. CECT 7805 was added as a suspension of living bacteria (LB), an autoclaved suspension (AS), and an extract obtained after sonication (ES) at different times (24, 48, and 72 h), to preformed biofilms of Mycobacterium abscessus DSM 44196, Mycobacterium chelonae ATCC 19235, and Mycobacterium fortuitum ATCC 6841, using a 96 h control of each species. The biofilms were analyzed by confocal laser scanning microscopy and by the Calgary biofilm device using the plates MBECTM Biofilm Inoculator. A statistically significant reduction in the thickness and covered surface was observed in all mycobacterial biofilms with all forms of Methylobacterium sp. A statistically significant increase in the autofluorescence was observed in M. abscessus biofilms but not in other biofilms. The increased percentage of dead mycobacteria was statistically significant in all cases. The reduced log CFU (colony-forming units)/peg recount was statistically significant in M. chelonae biofilms after treatment with AS and ES, but in M. fortuitum biofilms the recount decreased only with AS. M. abscessus biofilms were always significantly reduced with AS at 72 h and with ES. Methylobacterium sp. could inhibit RGM biofilm formation. Living cells of Methylobacterium sp. were not necessary to inhibit the growth of a preformed biofilm. M. chelonae biofilms were the most greatly reduced.


Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Methylobacterium/química , Mycobacterium/efeitos dos fármacos , Contagem de Colônia Microbiana , Testes de Sensibilidade Microbiana , Microscopia Confocal , Mycobacterium abscessus/efeitos dos fármacos
8.
J Antibiot (Tokyo) ; 72(12): 970-980, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31471594

RESUMO

Analogs of CPZEN-45, which is expected to be a promising new antituberculosis drug that overcomes the shortcomings of caprazamycins, were synthesized and their biological activities were evaluated. The biological activity of analogs 1-3, which converted the anilide portion, and analogs 4 and 5, focusing on the seven-membered ring, were lower than that of CPZEN-45. These results suggest that the inhibitory activity of CPZEN-45 against TagO, an ortholog of WecA, has a strict structural limitation, and it was hoped for elucidation of the mode of action of CPZEN-45 using structural biology in the future.


Assuntos
Antituberculosos/síntese química , Antituberculosos/farmacologia , Azepinas/química , Mycobacterium/efeitos dos fármacos , Relação Estrutura-Atividade , Antituberculosos/química , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Testes de Sensibilidade Microbiana , Estrutura Molecular , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/antagonistas & inibidores
9.
Eur J Med Chem ; 182: 111644, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31493745

RESUMO

The emergence of drug resistance in infectious microbial strains can be overcome by development of novel drug molecules against unexploited microbial target. The success of Bedaquiline in recent years, as FoF1 ATP synthase inhibitor against XDR and MDR mycobacterium strains, has resulted in further exploration to identify more potent and safe drug molecules against resistant strains. FoF1 ATP synthase is the main energy production enzyme in almost all eukaryotes and prokaryotes. Development of bacterial ATP synthase inhibitors is a safe approach, without causing harm to mammalian cells due to structural difference between bacterial and mammalian ATP synthase target sites. This review emphasizes on providing the structural insights for FoF1 ATP synthase of different prokaryotes and will help in the design of new potent antimicrobial agents with better efficacy. Further, applications of synthetic and natural active antimicrobial ATP synthase inhibitors, reported by different research groups are summarized. Their SAR and mode of actions are also analysed.


Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , Animais , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Relação Estrutura-Atividade
10.
J Microbiol Biotechnol ; 29(9): 1401-1411, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31434362

RESUMO

Mycobacterial cell walls comprise thick and diverse lipids and glycolipids that act as a permeability barrier to antibiotics or other chemical agents. The use of OH radicals from a non-thermal plasma jet (NTPJ) for the inactivation of mycobacteria in aqueous solution was adopted as a novel approach. Addition of water vapor in a nitrogen plasma jet generated OH radicals, which converted to hydrogen peroxide (H2O2) that inactivated non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis H37Rv. A stable plasma plume was obtained from a nitrogen plasma jet with 1.91 W of power, killing Escherichia coli and mycobacteria effectively, whereas addition of catalase decreased the effects of the former. Mycobacteria were more resistant than E. coli to NTPJ treatment. Plasma treatment enhanced intracellular ROS production and upregulation of genes related to ROS stress responses (thiolrelated oxidoreductases, such as SseA and DoxX, and ferric uptake regulator furA). Morphological changes of M. smegmatis and M. tuberculosis H37Rv were observed after 5 min treatment with N2+H2O plasma, but not of pre-incubated sample with catalase. This finding indicates that the bactericidal efficacy of NTPJ is related to the toxicity of OH and H2O2 radicals in cells. Therefore, our study suggests that NTPJ treatment may effectively control pulmonary infections caused by M. tuberculosis and nontuberculous mycobacteria (NTM) such as M. avium or M. abscessus in water.


Assuntos
Antibacterianos/farmacologia , Radical Hidroxila/farmacologia , Mycobacterium/efeitos dos fármacos , Gases em Plasma/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Meios de Cultura/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium/fisiologia , Nitrogênio/análise , Nitrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Gases em Plasma/química , Espécies Reativas de Oxigênio/metabolismo , Água/análise
11.
Tuberculosis (Edinb) ; 117: 45-51, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31378267

RESUMO

This manuscript reports, at the first time, the photoinactivation evaluation of tetra-cationic and anionic porphyrins as photosensitizers (PS) for the photodynamic inactivation (PDI) of rapidly growing mycobacteria strains. Two different charged porphyrin groups were obtained commercially. PDI experiments in the strains Mycobacterium massiliense e Mycobacterium fortuitum conducted with adequate concentration (without aggregation) of photosensitizer under white light at a fluence rate of 50 mW/cm2 over 90 min showed that the most effective PS caused a 100 times reduction in the concentration of viable mycobacteria. The present results show that porphyrin with positively charge are more efficient PS than anionic porphyrin (negatively charged) against M. massiliense e M. fortuitum. It is also clear that the effectiveness of the molecule as PS for PDI studies with mycobacteria is strongly related with the porphyrin peripheral charge, and consequently their solubility in physiological media. Cationic PSs might be promising anti-mycobacteria PDI agents with potential applications in medical clinical cases and bioremediation.


Assuntos
Mycobacterium/efeitos dos fármacos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Ânions , Cátions , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Humanos , Luz , Testes de Sensibilidade Microbiana/métodos , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Mycobacterium/fisiologia , Mycobacterium/efeitos da radiação , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium abscessus/fisiologia , Mycobacterium abscessus/efeitos da radiação , Mycobacterium fortuitum/efeitos dos fármacos , Mycobacterium fortuitum/fisiologia , Mycobacterium fortuitum/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo
12.
J Ethnopharmacol ; 244: 112095, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31325601

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Zingiber officinale (ginger) is a perennial herbaceous plant native in tropical Asia and generally cultivated in most American tropical countries with widespread use in popular medicine. Ginger essential oil (GEO) has been reported to exhibit several biological activities, such as antimicrobial. AIMS OF THE STUDY: The aim of this study was to determine the composition and the property of GEO and related fractions against Mtb and NTM, as well as their cytotoxicity. METHODS AND MATERIALS: GEO was obtained by hydrodistillation and fractionation was performed. Chemical characterization of GEO and fractions were carried out by gas chromatography/mass spectrometry. The antimycobacterial activity was evaluated by resazurin microtiter assay plate and broth microdilution method for Mtb and NTM, respectively. The cytotoxicity in Vero cells was assessed by MTT colorimetric assay. RESULTS: The analyses showed 63 compounds in the GEO sample, characterized by a high number of monoterpenes and sesquiterpenes. GEO fractionation rendered 11 fractions (FR1 to FR11). GEO and fractions minimum inhibitory concentration ranged from 31.25 to >250 µg/mL against Mtb and from 15.6 to >250 µg/mL against NTM. GEO showed better activity against NTM, M. chelonae, and M. abscessus sub. massiliense, than the semi-pure fractions. One fraction (FR5), containing γ-eudesmol as the main compound, was the most active against Mtb and NTM. The GEO and semi-pure fractions cytotoxicity assay showed CC50 63.3 µg/mL, and 36.3-312.5 µg/mL, respectively. CONCLUSIONS: In general, GEO showed a mix of monoterpenes and sesquiterpenes and a better antimycobacterial activity than the semi-pure fractions. Cytotoxic effects of GEO and its fractions should be better investigated.


Assuntos
Antibacterianos/farmacologia , Gengibre , Mycobacterium/efeitos dos fármacos , Óleos Voláteis/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Monoterpenos/farmacologia , Mycobacterium/crescimento & desenvolvimento , Rizoma , Sesquiterpenos/farmacologia , Células Vero
13.
ACS Chem Biol ; 14(7): 1490-1497, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31243958

RESUMO

Metabolic profiling and genome mining revealed that anaerobic bacteria have the potential to produce acyloin natural products. In addition to sattazolin A and B, three new sattazolin congeners and a novel acyloin named clostrocyloin were isolated from three strains of Clostridium beijerinckii, a bacterium used for industrial solvent production. Bioactivity profiling showed that the sattazolin derivatives possess antimicrobial activities against mycobacteria and pseudomonads with only low cytotoxicity. Clostrocyloin was found to be mainly active against fungi. The thiamine diphosphate (ThDP)-dependent sattazolin-producing synthase was identified in silico and characterized both in vivo and in in vitro enzyme assays. A related acyloin synthase from the clostrocyloin producer was shown to be responsible for the production of the acyloin core of clostrocyloin. The biotransformation experiments provided first insights into the substrate scope of the clostrocyloin synthase and revealed biosynthetic intermediates.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Bactérias Anaeróbias/química , Vias Biossintéticas , Clostridium/química , Hexanonas/química , Hexanonas/farmacologia , Humanos , Indóis/química , Indóis/farmacologia , Mycobacterium/efeitos dos fármacos , Infecções por Mycobacterium/tratamento farmacológico , Micoses/tratamento farmacológico , Pseudomonas/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico
14.
Arch Microbiol ; 201(9): 1195-1205, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31172252

RESUMO

Awareness of the consumer has increased the demand of safe and chemical-free foods, and consequently it has increased the demand of antibacterial bioactive compounds. In the present study, antibacterial compound produced by a local bacterial isolate NSD MTCC 10072, showing antagonistic activity against six human pathogens, was isolated, partially purified and characterized. Maximum production of antibacterial compound was observed between 51 and 60 h after seeding. The antibacterial activity of the compound was found to be thermostable up to 80 °C for 60 min and its efficacy was very good between pH 4 and 12. Minimum inhibitory concentration (25.84 µg/µl) of the antibacterial compound was observed against Streptococcus aureus NICM 2901. GC-MS analysis of the bacterium secreted chemical compound (C11H18N2O2) was used to identify the antimicrobial compound as Pyrrolo(1,2-a) pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl). In Silico studies showed that the antimicrobial compound is non-toxic, non-irritating and followed Lipinski-type properties which suggested that the compound could be used as potential drug against different human pathogens.


Assuntos
Antibacterianos/farmacologia , Bacillus cereus/metabolismo , Proteínas de Bactérias/farmacologia , Agentes de Controle Biológico/farmacologia , Antibacterianos/metabolismo , Bacillus cereus/isolamento & purificação , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Agentes de Controle Biológico/metabolismo , Escherichia coli/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium/efeitos dos fármacos , Pirróis/química , Pirróis/farmacologia , Staphylococcus aureus/efeitos dos fármacos
15.
Infect Genet Evol ; 73: 358-361, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31163274

RESUMO

BACKGROUND: Nontuberculous mycobacteria (NTM) are acid-fast bacilli (AFB) that can cause disease in human. Patients with NTM pulmonary disease can be falsely diagnosed with pulmonary tuberculosis (TB) due detection of AFB in sputum and similar clinical and chest X-ray picture. Laboratory detection of NTM is complicated and does not always mean presence of the disease, but can be attributed to colonization or sample contamination. Molecular tests, such as Genotype Mycobacterium CM/AS, allow quick and reliable detection of NTM. OBJECTIVE: To assess the NTM identification rate, to estimate the incidence of pulmonary NTM disease and to report the treatment outcomes among patients with NTM disease. DESIGN: Retrospective cohort design. RESULTS: NTM were detected among 92 (0.98 per 100,000 population) presumptive pulmonary TB patients in Arkhangelsk region in 2010-2017 among who 39 (0.42 per 100,000 population) patients were diagnosed with NTM disease. The most prevalent species found in our study were M. avium (33%) and M.intracellulare (11%). 69% of patients with NTM disease completed their treatment, 15% died, 13% were lost to follow up and 3% failed treatment. CONCLUSION: A system of diagnostics and treatment for NTM disease was set up in the Arkhangelsk region in Russia. Average NTM identification rate and incidence of pulmonary NTM disease were 0.98 per 100,000 and 0.42 per 100,000 population accordingly and were lower than reported in other studies. Treatment success rate in our study was 69% encouraging further improvements in diagnostics and treatment of patients with NTM.


Assuntos
Pneumopatias/diagnóstico , Pneumopatias/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Micobactérias não Tuberculosas/efeitos dos fármacos , Feminino , Humanos , Incidência , Pulmão/efeitos dos fármacos , Pneumopatias/microbiologia , Masculino , Pessoa de Meia-Idade , Mycobacterium/efeitos dos fármacos , Estudos Retrospectivos , Federação Russa , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologia
16.
Int J Med Microbiol ; 309(5): 319-324, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31138496

RESUMO

Cell wall biosynthesis represents a valid target for antibacterial action but only a limited number of chemical structure classes selectively interact with specific enzymes or protein structures like transporters of the cell envelope. The integral membrane protein MraY translocase is essential for peptidoglycan biosynthesis catalysing the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate, thereby generating the cell wall intermediate lipid I. Not present in eukaryotic cells, MraY is a member of the superfamily of yet not well-understood integral membrane enzymes which involve proteins for bacterial lipopolysaccharide and teichoic acid or eukaryotic N-linked saccharides biosynthesis. Different natural nucleoside antibiotics as inhibitors of MraY translocase have been discovered comprising a glycosylated heterocyclic pyrimidin base among other potential lipid-, peptidic- or sugar moieties. Caprazamycins are liponucleoside antibiotics isolated from Streptomyces sp. MK730-62F2. They possess activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including M. intracellulare, M. avium and M. tuberculosis. Structural elucidation revealed the (+)-caprazol core skeleton as a unique moiety, the caprazamycins share with other MraY inhibitors such as the liposidomycins, A-90289 and the muraminomicins. They also share structural features such as uridyl-, aminoribosyl- and fatty acyl-moieties with other MraY translocase inhibitors like FR-900493 and the muraymycins. Intensive studies on their biosynthesis during the last decade identified not only common initial biosynthetic steps, but also revealed possible branching points towards individual biosynthesis of the respective compound. Structural diversity of caprazamycins was generated by feeding experiments, genetic engineering of the biosynthetic gene clusters and chemical synthesis for structure activity relationship studies with its target, MraY translocase.


Assuntos
Antibacterianos/química , Azepinas/química , Proteínas de Bactérias/antagonistas & inibidores , Nucleosídeos/química , Streptomyces/química , Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Vias Biossintéticas , Estrutura Molecular , Família Multigênica , Mycobacterium/efeitos dos fármacos , Relação Estrutura-Atividade
17.
Infect Genet Evol ; 73: 132-138, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31048076

RESUMO

The 2nd St. Petersburg Symposium on Tuberculosis and Mycobacteria: Molecular Approach, was held in St. Petersburg, Russia on 5-6 December 2018. A special issue of Infection, Genetics and Evolution will publish articles based on the selected presentations. In this paper, I will discuss some of the hot topics of molecular mycobacteriology highlighted at this meeting that I had the pleasure to organize and honor to chair. The symposium addressed interrelated fundamental and applied issues of modern mycobacteriology such as molecular evolution and phylogenomics, host-microbe interactions and pathogenesis, coevolution of M. tuberculosis with humans, new genomic and postgenomic technologies. Molecular methods for TB diagnostics and drug resistance detection are supported by WHO and whole genome/next generation sequencing presents a comprehensive approach. At the same time, cost and implementation of new methods for direct analysis of clinical samples and/or in low-resource settings remain a great challenge. A due attention was also given to the medically important nontuberculous mycobacteria. Assessment of spectrum of the circulating mycobacterial species in the Russian Federation and the countries of the European Union was presented and the underlying reasons of the observed diversity were discussed. To conclude, the symposium became a multidisciplinary event that was useful to promote networking and exchange of knowledge and experience. The next (third) symposium was planned to be organized in 2021.


Assuntos
Mycobacterium/genética , Tuberculose/microbiologia , Evolução Molecular , Genoma Bacteriano , Genômica/métodos , Humanos , Técnicas de Diagnóstico Molecular , Epidemiologia Molecular , Mycobacterium/classificação , Mycobacterium/efeitos dos fármacos , Mycobacterium tuberculosis , Micobactérias não Tuberculosas , Tuberculose/diagnóstico , Tuberculose/tratamento farmacológico , Tuberculose/epidemiologia
18.
J Vis Exp ; (146)2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-31081825

RESUMO

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.


Assuntos
Genes Reporter , Mycobacterium/genética , Biossíntese de Proteínas , Aminoglicosídeos/farmacologia , Códon/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Fluorescência Verde/metabolismo , Luciferases de Vaga-Lume/genética , Mycobacterium/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos
19.
Methods Mol Biol ; 1964: 141-149, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30929241

RESUMO

Measuring metabolic activity and response of biofilm to different conditions or compounds is of general interest but is also expected to help in developing new antibiofilm compounds and potentially new treatments. Current culture-based and microscopic methods although of much use have several drawbacks. Isothermal calorimetry can be useful in this context by allowing measurements of the metabolic activity of biofilm grown and maintained on solid medium. Biofilms prepared on membranes were placed in calorimetry vials containing solid medium. Sealed vials were introduced in an isothermal calorimeter, and the rate of metabolic heat production was monitored over time. We chose mycobacteria as an example for this paper as working with mycobacterial biofilms is notoriously difficult.


Assuntos
Biofilmes/efeitos dos fármacos , Calorimetria/métodos , Mycobacterium/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Mycobacterium/crescimento & desenvolvimento
20.
Biochim Biophys Acta Biomembr ; 1861(6): 1213-1227, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31002767

RESUMO

Lipid structure critically dictates the molecular interactions of drugs with membranes influencing passive diffusion, drug partitioning and accumulation, thereby underpinning a lipid-composition specific interplay. Spurring selective passive drug diffusion and uptake through membranes is an obvious solution to combat growing antibiotic resistance with minimized toxicities. However, the spectrum of complex mycobacterial lipids and lack thereof of suitable membrane platforms limits the understanding of mechanisms underlying drug-membrane interactions in tuberculosis. Herein, we developed membrane scaffolds specific to mycobacterial outer membrane and demonstrate them as improvised research platforms for investigating anti-tubercular drug interactions. Combined spectroscopy and microscopy results reveal an enhanced partitioning of model drug Rifabutin in trehalose dimycolate-containing mycobacterial membrane systems. These effects are apportioned to specific changes in membrane structure, order and fluidity leading to enhanced drug interaction. These findings on the membrane biophysical consequences of drug interactions will offer valuable insights for guiding the design of more effective antibiotic drugs coupled with tuned toxicity profiles.


Assuntos
Antituberculosos/farmacologia , Bicamadas Lipídicas/metabolismo , Modelos Biológicos , Mycobacterium/metabolismo , Rifabutina/farmacologia , Fenômenos Biofísicos , Avaliação Pré-Clínica de Medicamentos , Fluidez de Membrana , Mycobacterium/efeitos dos fármacos , Temperatura
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