Mutations related to streptomycin resistance in the rpsL and rrs genes are well known and can explain about 70% of this phenotypic resistance. Recently, the gidB gene was found to be associated with low-level streptomycin resistance in Mycobacterium tuberculosis. Mutations in gidB have been reported with high frequency, and this gene appears to be very polymorphic, with frameshift and point mutations occurring in streptomycinsusceptible and streptomycin-resistant strains. In this study, mutations in gidB appeared in 27% of streptomycin-resistant strains that contained no mutations in the rpsL or rrs genes, and they were associated with low-level streptomycin resistance. However, the association of certain mutations in gidB with streptomycin resistance needs to be further investigated, as we also found mutations in gidB in streptomycin-susceptible strains. This occurred only when the strain was resistant to rifampin and isoniazid. Two specific mutations appeared very frequently in this and other studies of streptomycin-susceptible and -resistant strains; these mutations were not considered related to streptomycin resistance, but as a polymorphism. We stratified the strains according to the different phylogenetic lineages and showed that the gidB 16 polymorphism (16G allele) was exclusively present in the Latin American-Mediterranean (LAM) genotype, while the gidB 92 polymorphism (92C allele) was associated with the Beijing lineage in another population. In the sample studied, the two characterized single-nucleotide polymorphisms could distinguish LAM and Beijing lineages from the other lineages.

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates.

Bovine tuberculosis (BTB) remains an important economic and zoonotic problem in Latin America. Traditionally, the fight against BTB is initiated by the implementation of routine diagnostic tests for certification of free properties. The diagnosis of BTB can be made by direct and indirect methods, in which we can mention clinical, post mortem, histopathological, immunological, bacteriological and molecular methods. The renewal of scientific interest in tuberculosis in recent year has led to develop and improve methods of diagnosis, prevention, control and eradication of BTB. The aim of this review is to present and discuss different diagnosis methods of BTB.Keywords: tuberculosis bovine, diagnosis, Mycobacterium bovis, new techniques. Diagnóstico de tuberculose bovina: revisão das principais técnicas ResumoA tuberculose bovina (BTB) continua sendo um importante problema econômico na América Latina, com potenciais consequências zoonóticas. Tradicionalmente, a luta contra a tuberculose bovina tem sido iniciada pela execução de testes de diagnóstico de rotina para a certificação de propriedades livres da doença. O diagnóstico de BTB pode ser feito através de métodos diretos e indiretos, nos quais podemos citar os métodos clínicos, post mortem, histopatológicos, imunológicos, bacteriológicos e moleculares. A renovação do interesse científico em tuberculose nos últimos anos tem levado à necessidade de desenvolver e melhorar os métodos de diagnóstico, prevenção, controle e erradicação da BTB. O objetivo deste artigo é analisar e discutir sobre os diferentes métodos de diagnóstico de BTB.Palavras-chave: tuberculose bovina, diagnóstico, Mycobacterium bovis, novas técnicas.

Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality that is often disregarded by the tuberculosis specialists in favor of the almost undisputed importance of antibiotic target-gene mutations for the resistance in M. tuberculosis.

Duroia macrophylla popularly known as “cabeça-de-urubú,” “apuruí,” or “puruí-grande-da-mata” occurs in the Amazon Forest. Its leaves and branches were collected twice and extracted with dichloromethane and methanol. All extracts were subjected to phytochemical investigation and terpenes and flavonoids were found in all dichloromethane and methanol extracts, respectively. Methanol extracts from both branches (1st collection) and leaves (2nd collection) presented hydrolyzed tannins, yet alkaloids were only detected in the dichloromethane and methanol extracts from branches at the 2nd collection. Phenol compounds were found in both dichloromethane extracts' collections. The action of every extract was assayed against Mycobacterium tuberculosis (RMPr, H37Rv, and INHr strains), showing that the dichloromethane extract from leaves (1st collection) has the major biological activity, with a MIC of 6.25 μg/mL for the INHr strain, 25.0 μg/mL for the RMPr strain, and ≤6.25 μg/mL for the H37Rv strain. The chromatographic fractioning of the dichloromethane extract from leaves (1st collection) yielded the isolation of two triterpenes: oleanolic and ursolic acids, which were identified by NMR analysis and reported for the first time in the Duroia genus.