Essential oil of Gallesia integrifolia is active against mycobacteria.

Background: There is critical need for new therapeutic options for treatment of diseases caused by mycobacteria. Materials & methods: Gallesia integrifolia essential oils (EOs) and crude extracts (CEs) were tested for their anti-Mycobacterium tuberculosis and anti-nontuberculous mycobacteria activity. Results: Minimum inhibitory concentration (MIC) of EOs ranged from 15.63 to 62.5 µg/ml against M. tuberculosis and 62.5 to >250 µg/ml against nontuberculous mycobacteria. CEs showed low activity. All EO tested demonstrated synergism with antituberculosis drugs. The cytotoxicity of EOs and CEs, in different cell lines, showed selectivity index from 2.2 to 9.8 and >0.056 to 2.0, respectively. Conclusion: G. integrifolia EOs are a candidate for the development of new therapeutic options in the treatment of tuberculosis and other mycobacterial diseases.

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.

Proteomic profiling of Klebsiella pneumoniae carbapenemase (KPC)-producer Klebsiella pneumoniae after induced polymyxin resistance.

Aim: To elucidate the changes in protein expression associated with polymyxin resistance in Klebsiella pneumoniae, we profiled a comparative proteomic analysis of polymyxin B-resistant mutants KPC-2-producing K. pneumoniae, and of its susceptible counterparts. Material & methods: Two-dimensional reversed phase nano ultra-performance liquid chromatography mass spectrometry was used for proteomic analysis. Results: Our results showed that the proteomic profile involved several biological processes, and we highlight the downregulation of outer membrane protein A (OmpA) and the upregulation of SlyB outer membrane lipoprotein (conserved protein member of the PhoPQ regulon) and AcrA multidrug efflux pump in polymyxin B-resistant strains. Conclusion: Our results highlight the possible participation of the SlyB, AcrA and OmpA proteins in the determination of polymyxin B heteroresistance in KPC-2-producing K. pneumoniae.

Structure-activity relationship of natural and synthetic coumarin derivatives against Mycobacterium tuberculosis.

Aim: Eight coumarin derivatives (1a-h) obtained from natural (-)-mammea A/BB (1) and 13 synthetic coumarins (2-14) had their cytotoxicity and biological activity evaluated against Mycobacterium tuberculosis H37Rv reference strain and multidrug-resistant clinical isolates. Materials & methods: Anti-M. tuberculosis activity was evaluated by resazurin microtiter assay plate, and the cytotoxicity of natural and synthetic products using J774A.1 macrophages by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Results: Compounds 1g, 5, 6, 12 and 14 were more active against M. tuberculosis H37Rv and multidrug-resistant clinical isolates with MIC values ranging from 15.6 to 62.5 µg/ml. Conclusion: These results demonstrate that the coumarin derivatives were active against multidrug-resistant clinical isolates, becoming potential candidates to be used in the treatment of resistant tuberculosis.

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.

Novel 4-methoxynaphthalene-N-acylhydrazones as potential for paracoccidioidomycosis and tuberculosis co-infection.

Aim: 17 new 4-methoxynaphthalene-N-acylhydrazones were synthesized in order to evaluate their biological action against important pathogens. Methods: In vitro susceptibility assays of compounds were performed against Paracoccidioidesbrasiliensis and Mycobacterium tuberculosis. Results: Compounds 4a, 4b and 4k were the most potent against P. brasiliensis, two with minimum inhibitory concentrations of ≤1 µg ml-1 and exhibited pharmacological synergy with amphotericin B. The compounds also showed activity against M. tuberculosis, with 4c and 4k being the more promising. Compound 4k showed good synergistic antimycobacterium activity with ethambutol. None of the compounds tested showed toxicity. Conclusion: We highlight the compound 4k, as a potential agent for the treatment of patients co-infected with paracoccidioidomycosis and tuberculosis.

Modulatory effects of verapamil in rifampicin activity against Mycobacterium tuberculosis.

AIM: To evaluate modulatory effect of verapamil (VP) in rifampicin (RIF) activity and its effect in efflux pumps (EPs) transcript levels in Mycobacterium tuberculosis. MATERIALS & METHODS: RIF and VP minimal inhibitory concentration, combinatory effect and detection of mutations were determined in 16 isolates. EPs transcript levels were determined in four isolates by real-time PCR after exposure to drugs. RESULTS: VP showed good combinatory effect among RIF-resistant isolates. This effect was also observed in the relative transcript levels of EPs, mainly after 72 h of exposure, depending on the EP gene, genotype and the resistance profile of the isolate. CONCLUSION: Additional regulatory mechanisms in the EP activities, as well as, interactions with other drug-specific resistance mechanisms need further investigation in M. tuberculosis.

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.

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

AIM: We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. METHODS: The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. RESULTS: EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. CONCLUSION: Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Proteomic and morphological changes produced by subinhibitory concentration of isoniazid in Mycobacterium tuberculosis.

AIM: To study the proteomic and morphological changes in Mycobacterium tuberculosis H37Rv exposed to subinhibitory concentration of isoniazid (INH). MATERIALS & METHODS: The bacillus was exposed to ½ MIC of INH at 12, 24 and 48 h. The samples' cells were submitted to scanning electron microscopy. The proteins were separated by 2D gel electrophoresis and identified by MS. RESULTS: INH exposure was able to alter the format, the multiplication and causing a cell swelling in the bacillus. The major altered proteins were related to the virulence, detoxification, adaptation, intermediary metabolism and lipid metabolism. CONCLUSION: The protein and morphological changes in M. tuberculosis induced by ½ MIC INH were related to defense mechanism of the bacillus or the action of INH therein.