Is the efflux pump inhibitor Verapamil a potential booster for isoniazid against Mycobacterium tuberculosis?

The membrane-based efflux pump systems are recognized to have an important role in pathogenicity and drug resistance in Mycobacterium tuberculosis by the extrusion of toxic substrates and drugs from the inner bacillus. This study aimed to investigate the in vitro interaction of Verapamil (VP), an efflux pump inhibitor, with the classical first-line anti-tuberculosis drug isoniazid (INH) in resistant and susceptible M. tuberculosis clinical isolates. Seven multidrug-resistant (MDR), three INH monoresistant and four susceptible M. tuberculosis clinical isolates were tested for the INH and VP combination by modified Resazurin Microtiter Assay Plate (REMA). Fractional Inhibitory Concentration (FIC) and Modulation Factor (MF) were determined. The INH plus VP combination showed no significant change in the Minimum inhibitory concentration (MIC) values of INH (FIC≥ 0.5; MF=1 or 2).The use of VP in tuberculosis therapy should be managed carefully, considering the resistance caused by specific mutation in katG and inhA genes, in which the use of these EPIs may have no success. The use of EPIs as an adjunctive drug in the anti-tuberculosis therapy should be further investigated on a larger number of M. tuberculosis clinical isolates with different resistant profile.

Insulin-loaded polymeric mucoadhesive nanoparticles: development, characterization and cytotoxicity evaluation

Abstract Mucoadhesive nanoparticles are particularly interesting for delivery through nasal or pulmonary routes, as an approach to overcome the mucociliary clearance. Moreover, these nanoparticles are attractive for peptide and protein delivery, particularly for insulin to treat diabetes, as an alternative to conventional parenteral administration. Thus, chitosan, a cationic mucoadhesive polysaccharide found in shells of crustaceans, and the negatively-charged dextran sulfate are able to form nanoparticles through ionic condensation, representing a potential insulin carrier. Herein, chitosan/dextran sulfate nanoparticles at various ratios were prepared for insulin loading. Formulations were characterized for particle size, zeta potential, encapsulation efficiency, scanning electron microscopy, differential scanning calorimetry, and in vitro drug release. Moreover, the interaction with mucin and the cytotoxicity against a lung cell line were studied, which altogether have not been addressed before. Results evidenced that a proper selection of polyelectrolytes is necessary for smaller particle size formation and also the composition and zeta potential impact encapsulation efficiency, which is benefited by the positive charge of chitosan. Insulin remained stable after encapsulation as evidenced by calorimetric assays, and was released in a sustained manner in the first 10 h. Positively-charged nanoparticles based on chitosan/dextran-sulfate at the ratio of 6:4 successfully interacted with mucin, which is a prerequisite for delivery to mucus-containing tissues. Finally, insulin-loaded nanoparticles displayed no cytotoxicity effect against lung cells at tested concentrations, suggesting the potential for further in vivo studies.

Molecular characterization of Mycobacterium tuberculosis and Mycobacterium bovis isolates by Enterobacterial Repetitive Intergenic Consensus-PCR

Tuberculosis (TB) is an infectious disease in which the molecular typing methods allow to have important information about the dynamics of transmission and to assist properly in disease control. Although the ERIC-PCR (Enterobacterial repetitive intergenic consensus-PCR) assay is fast and easy to perform, scarce studies have reported its use in epidemiological studies in TB outbreaks. In this study, we aimed to genotype Mycobacterium tuberculosis and M. bovis isolates by ERIC-PCR and compare its discriminatory power with two other classically used methods: 12 loci-MIRU (Mycobacterial Interspersed Repetitive Units) and Spoligotyping. The M. tuberculosis isolates studied were from northwestern and southwestern and M. bovis from northwestern Parana, Brazil. ERIC-PCR rendered banding patterns with great diversity (1 to 12 bands) of molecular sizes, ranging from 100 to 1600 bp. ERIC-PCR showed to be fast, simple and affordable to differentiate isolates. ERIC-PCR would be an important tool in the epidemiology of TB as screening in case of outbreak, which demands rapid intervention. However if any doubt persist, as it may occur with the application of only one genotypic method, other genotyping methods should be applied and carefully interpreted, always with additional epidemiological information.

Esteroide e Triterpenos de espécies de Qualea - Bioatividade sobre Mycobacterium tuberculosis / Sterol and Triterpenes from Qualea species: Bioactivity on Mycobacterium tuberculosis

O extrato clorofórmico das cascas de Qualea parviflora (Vochysiaceae) foi fracionado em cromatografia em coluna usando sílica gel para fornecer triterpenos (lupeol, lupenona, betulina, ácido epibetulínico, e friedelina) e um esteroide (β-sitosterol). Os compostos β- sitosterol, lupenona e lupeol foram identificados também em Q. grandiflora e Q. multiflora, enquanto friedelina foi detectada apenas em Q. multiflorautilizando a cromatografia gasosa acoplada à espectrometria de massas. A atividade anti Mycobacterium tuberculosis do extrato clorofórmico e dos compostos isolados foi determinada por MABA e o valor da CIM variou de 250,0 a 31,2 μg/mL. Esta investigação constitui o primeiro relato do estudo químico e antitubercular de compostos apolares das espécies de Qualea.

The chloroform extract of bark of the tropical tree Qualea parviflora (Vochysiaceae) was fractionated by column chromatography on silica gel, yielding triterpenes (lupeol, lupenone, betulin, epibetulinic acid and friedelin) and a steroid (β-sitosterol). β-sitosterol, lupenone and lupeol were also identified in Q. grandiflora and Q. multiflora, while friedelin was detected only in Q. Multiflora, by means of gas chromatography-mass spectrometry. The anti-Mycobacterium tuberculosis activity of the chloroform extract and isolated compounds was assayed by MABA and MIC values ranged from 250.0 to 31.2 μg/mL. This study is the first to investigate the chemistry and antitubercular activity of apolar compounds from Qualeaspecies.

Phenolic compounds and antioxidant, antimicrobial and antimycobacterial activities of Serjania erecta Radlk. (Sapindaceae)

Serjania erecta Radlk.(Sapindaceae) is a medicinal plant traditionally used in Brazil. We assayed the ethanolic extract of leaves and roots against seven microorganisms. The REMA (Resazurin Microtiter Assay) assay was used to measure the biological activity in vitro against Mycobacterium tuberculosis and our results showed moderate activity of the ethanolic extract. On the other hand S. aureus, P. aeruginosa, S. setubal, C. albicans, S. cerevisiae and E. coli revealed that the leaves and roots of S. erecta inhibited the growth of all microorganisms. The ethanolic extracts of leaves and roots showed low values of antioxidant activities. The ethanolic extracts of leaves and roots were analyzed by chromatographic and spectrometric methods. (-)-Epicatechin, kaempferol aglycone and five glycoside derivates were isolated: kaempferol-3-O-α-L-rhamnopyranoside, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside from the roots and kaempferol, kaempferol 3,7-di-O-α-L-rhamnopyranoside, vitexin, isovitexin and (-)-epicatechin in the leaves. This is the first chemical study reported in the literature about this specie.

Serjania erecta Radlk. (Sapindaceae) é uma planta medicinal utilizada no Brasil. Analisamos o extrato etanólico de folhas e raízes para sete microorganismos. O REMA (Ensaio de Microtitulação Resazurina) foi utilizado para medir a atividade biológica in vitro contra o Mycobacterium tuberculosis e nossos resultados mostraram atividade moderada do extrato etanólico. Por outro lado, ensaios com S. aureus, P. aeruginosa, S. setubal, C. albicans, S. cerevisiae e E. coli revelaram que as folhas e raízes de S. erecta inibiram crescimento em todos os microorganismos. Os extratos etanólicos de folhas e raízes apresentaram valores baixos de atividade antioxidante. Os extratos etanólicos de folhas e raízes foram analisados por métodos cromatográficos e espectroscópicos. (-)-Epicatequina, canferol aglicona e cinco derivados glicosídicos foram isolados: canferol -3-O-α-L-ramnopiranosídeo, canferol-3-O-α-L-ramnopiranosil-(1→6)-β-D-glucopiranosídeo a partir das raízes e canferol, canferol 3,7-di-O-α-L-ramnopiranosídeo, vitexina, isovitexina e (-) epicatequina nas folhas. Este é o primeiro estudo químico na literatura sobre esta espécie.

Comparison of resazurin microtiter assay performance and BACTEC MGIT 960 in the susceptibility testing of Brazilian clinical isolates of Mycobacterium tuberculosis to four first-line drugs

We assessed the performance of REMA in comparison with BACTEC MGIT 960 in the susceptibility testing of 80 Mycobacterium tuberculosis clinical isolates from Clemente Ferreira Institute against four drugs. REMA proved to be a rapid and accurate method, providing excellent correlation with BACTEC MGIT 960, with the exception of results for the ethambutol drug.

Anti-Mycobacterium tuberculosis activity of fungus Phomopsis stipata

Our purpose was to determine the anti-Mycobacterium tuberculosis activity of the metabolites produced by the endophitic fungus Phomopsis stipata (Lib.) B. Sutton, (Diaporthaceae), cultivated in different media. The antimycobacterial activity was assessed through the Resazurin Microtiter Assay (REMA) and the cytotoxicity test performed on macrophage cell line. The extracts derived from fungi grown on Corn Medium and Potato Dextrose Broth presented the smallest values of Minimum Inhibitory Concentration (MIC) and low cytotoxicity, which implies a high selectivity index. This is the first report on the chemical composition and antitubercular activity of metabolites of P. stipata, as well as the influence of culture medium on these properties.