Rescue of susceptibility to second-line drugs in resistant clinical isolates of Mycobacterium tuberculosis.

Aim: Antibiotic resistance is one of the biggest threats to global health, and this study aimed better understand how the efflux pumps are related to this process in tuberculosis clinical isolates. Results: The combination of antibiotics plus efflux pumps (EP) inhibitors was able to restore the susceptibility of clinical isolates in 100% of aminoglycosides resistance and 33.3% of the fluoroquinolones resistance. The relative expression of EP genes in pre-extensively drug-resistant isolates showed an increase of up to 1000-times. Conclusion: The rescue of susceptibility in the presence of EP inhibitors, the increased of activity and expression of the EP genes alert that the inhibition of EP can reduce the selection of resistant strains and improve treatment.

3,5-dinitrobenzoylhydrazone derivatives as a scaffold for antituberculosis drug development.

Background: The development of drugs is essential to eradicate tuberculosis. Materials & methods: Sixteen 3,5-dinitrobenzoylhydrazone (2-17) derivatives and their synthetic precursors 3,5-dinitrobenzoylhydrazide (1) and methyl ester (18) were screened for their anti-Mycobacterium tuberculosis (Mtb) potential. Results: Twelve compounds had minimum inhibitory concentration (MIC) ranging from 0.24 to 7.8 µg/ml against the Mtb strain. The activity was maintained in multidrug-resistant Mtb clinical isolates. Only compound (17) showed activity against nontuberculous mycobacteria. The compounds exhibited a limited spectrum of activity, with an MIC >500 µg/ml against Gram-positive and -negative bacteria. Compounds (2), (5) and (11) showed a synergistic effect with rifampicin. An excellent selectivity index value was found, with values reaching 583.33. Conclusion: 3,5-dinitrobenzoylhydrazone derivatives could be considered as a scaffold for the development of antituberculosis drugs.

(-)-Camphene-based derivatives as potential antibacterial agents against Staphylococcus aureus and Enterococcus spp.

Aim: To evaluate the activity of (-)-camphene-based thiosemicarbazide (TSC) and 4-hydroxy-thiosemicarbazone (4-OH-TSZ), alone and in combination against Gram-positive. Material & methods: MIC were determined for Staphylococcus aureus, Enterococcus spp. reference strains and clinical isolates. Drug combination, time-kill and cytotoxicity assays were also performed. Results: TSC and 4-OH-TSZ demonstrated potent inhibitory activity against S. aureus and Enterococcus spp., including multidrug-resistant isolates (MIC ranging from 1.9 to 31.2 µg/ml), and were bactericidal for the reference strains of both Gram-positive tested. The derivatives proved to be selective for the bacteria and synergistic with oxacillin and vancomycin. Conclusion: (-)-Camphene-based derivatives can represent promising drug candidates against critical pathogens, such as S. aureus and Enterococcus spp., including MRSA and vancomycin resistance Enterococcus spp. isolates.

Synthesis and biological evaluation of 12 novel (-)-camphene-based 1,3,4-thiadiazoles against Mycobacterium tuberculosis.

Aim: To evaluate the activity, cytotoxicity and efflux pumps inhibition of a series of 12 novels (-)-camphene-based 1,3,4-thiadiazoles (TDZs) against Mycobacterium tuberculosis (Mtb). Materials & methods: The minimum inhibitory concentration (MIC), cytotoxicity for three cell lines, ethidium bromide accumulation and checkerboard methods were carried out. Results: Compounds (6a, 6b, 6c, 6g, 6h and 6j) showed significant anti-Mtb activity (MIC 3.9-7.8 µg/ml) and no antagonism with anti-TB drugs already used in the TB treatment. Selectivity index (SI) was also determined, with values reaching 42.9 for H37Rv strain and 97.1 for clinical isolate. Five compounds also showed bacterial efflux pumps inhibition and one showed modulator effect with three drugs. Conclusion: These six TDZs should be considered as new scaffolds to develop anti-TB drugs.

Hydrazone, benzohydrazones and isoniazid-acylhydrazones as potential antituberculosis agents.

Aim: To evaluate the potential of three benzohydrazones (1-3), four acylhydrazones derived from isoniazid (INH-acylhydrazones) (4-7) and one hydrazone (8) as antituberculosis agents. Materials & methods: Inhibitory and bactericidal activities were determined for the reference Mycobacterium tuberculosis (Mtb) strain and clinical isolates. Cytotoxicity, drug combinations and ethidium bromide accumulation assays were also performed. Results: The tested compounds (1-8) presented excellent antituberculosis activity with surprisingly inhibitory (0.12-250 µg/ml) and bactericidal values, even against multidrug-resistant Mtb clinical isolates. Compounds showed high selectivity index, with values reaching 1833.33, and a limited spectrum of activity. Some of the compounds (2 & 8) are also great inhibitors of bacillus efflux pumps. Conclusion: Benzohydrazones and INH-acylhydrazones may be considered scaffolds for the development of new anti-Mtb drugs.

Eugenol and derivatives activity against Mycobacterium tuberculosis, nontuberculous mycobacteria and other bacteria.

AIM: To evaluate (i) the in vitro activity of eugenol (EUG) and three derivatives against Mycobacterium tuberculosis (Mtb), nontuberculous mycobacteria (NTM) and other bacteria, (ii) the EUG and antituberculosis drugs combinatory effect and (iii) the EUG and its derivatives cytotoxicity. MATERIALS & METHODS: Minimum inhibitory concentration of the compounds were determined by resazurin microtiter or broth microdilution assay and the drug interaction between EUG and antituberculosis drugs by resazurin drug combination microtiter. The cytotoxicity was carried out in macrophages, HeLa and VERO cells. Results: EUG and derivatives displayed activity and synergic effect of EUG combined with rifampicin, isoniazid, ethambutol, and pyrazinamide in Mtb including multidrug-resistant isolates, with more selectivity to bacillus than macrophages, HeLa and VERO cells (selective index from 0.65 to 31.4). EUG derivatives (4-allyl-2-methoxyphenyl acetate, 4-allyl-2-methoxyphenyl benzoate, and 4-allyl-2-methoxyphenyl 4-nitrobenzoate) were more active against nontuberculous mycobacteria than EUG. EUG and derivatives exhibited low activity in other Gram-positive and -negative bacteria. CONCLUSION: EUG and its derivatives show activity against Mycobacterium spp. and synergic effect of EUG combined with antituberculosis drugs against Mtb.

New insights on Ethambutol Targets in Mycobacterium tuberculosis.

BACKGROUND: In recent years, very few effective drugs against Mycobacterium tuberculosis have emerged, which motivates the research with drugs already used in the treatment of tuberculosis. Ethambutol is a bacteriostatic drug that affects cell wall integrity, but the effects of this drug on bacilli are not fully exploited. OBJECTIVE: Based on the need to better investigate the complex mechanism of action of ethambutol, our study presented the proteome profile of M. tuberculosis after different times of ethambutol exposure, aiming to comprehend the dynamics of bacilli response to its effects. M. tuberculosis was exposed to ½ MIC of ethambutol at 24 and 48 hours. The proteins were identified by MALDI-TOF/TOF. RESULTS: The main protein changes occurred in metabolic proteins as dihydrolipoyl dehydrogenase (Rv0462), glutamine synthetase1 (Rv2220), electron transfer flavoprotein subunit beta (Rv3029c) and adenosylhomocysteinase (Rv3248c). CONCLUSION: Considering the functions of these proteins, our results support that the intermediary metabolism and respiration were affected by ethambutol and this disturbance provided proteins that could be explored as additional targets for this drug.

Synthesis of novel (-)-Camphene-based thiosemicarbazones and evaluation of anti-Mycobacterium tuberculosis activity.

In this work the aim of study was the synthesis and evaluation of in vitro anti-Mycobacterium tuberculosis activity as well as the cytotoxicity in VERO cells of a series of 17 novel thiosemicarbazones derived from the natural monoterpene (-)-camphene by REMA and MTT methods. Overall, the majority of tested compounds exhibited considerable inhibitory effects on the growth of M. tuberculosis H37Rv, especially the derivatives 3, 4a-c, 4f, 4i, 4k, 5 and 6a-b. MIC values of 20 tested compounds ranged from 3.9 to > 250 µg/mL. It was found that when inserting new nitrogenous groups to the (-)-camphene increased the anti-M. tuberculosis activity of some compounds. The SI was calculated for all compounds that showed highly potent anti-M. tuberculosis activity and the best SI values were 21.36, 26.92 and 31.62 (4b, 6a and 6b), and may be considered potential candidates for future antituberculosis drugs.

Carvacrol activity & morphological changes in Mycobacterium tuberculosis.

AIM: Evaluating carvacrol, derivatives and carvacrol plus anti-TB (anti-tuberculous) drug combination activities in Mycobacterium tuberculosis as well as carvacrol cytotoxicity, efflux pump inhibitor activity and morphological changes in M. tuberculosis H37Rv. METHODS: Carvacrol (CAR) and derivatives' activities were determined by resazurin microtiter assay and drug interaction by resazurin drug combination microtiter. Carvacrol cytotoxicity in VERO cells and efflux pumps inhibitor activity by ethidium bromide assay were determined and scanning electron microscopy performed. RESULTS: Carvacrol MIC ranged from 19 to 156 µg/ml and carvacrol plus rifampicin combination showed synergistic effect in clinical isolates. No anti-M. tuberculosis activity improvement was observed with carvacrol derivatives. Carvacrol showed to be selective for M. tuberculosis, to have efflux pumps activity and to induce rough bacillary and agglomerates. CONCLUSION: Carvacrol shows good anti-M. tuberculosis activity and synergism with rifampicin.