Resazurin Microtiter Assay Plate Research Articles

Optimization of the biosynthesis of silver nanoparticles using bacterial extracts and their antimicrobial potential

In the present study, silver nanoparticles (AgNPs) were biosynthesized using the supernatant and the intracellular extract of Cupriavidus necator, Bacillus megaterium, and Bacillus subtilis. The characterization of the AgNPs was carried out using UV–Vis spectroscopy, FTIR, DLS and TEM. Resazurin microtiter-plate assay was used to determine the antimicrobial action of AgNPs against Escherichia coli. UV–Visible spectra showed peaks between 414 and 460 nm. TEM analysis revealed that the synthesized AgNPs showed mostly spherical shapes. DLS results determined sizes from 20.8 to 118.4 nm. The highest antimicrobial activity was obtained with the AgNPs synthesized with supernatant rather than those using the intracellular extract. Therefore, it was determined that the bacterial species, temperature, pH, and type of extract (supernatant or intracellular) influence the biosynthesis. This synthesis thus offers a simple, environmentally friendly, and low-cost method for the production of AgNPs, which can be used as antibacterial agents.

Hedychium coronarium J.Koenig produced extracellular and intracellular anti-tuberculosis activity with morphological alteration of the tubercle bacillus

Hedychium coronarium J.Koenig (Zingiberaceae) is a medium-sized aromatic rhizomatous perennial monocotyledonous plant. The rhizome of H. coronarium is reported in traditional medicine for its use in the treatment of tonsillitis, bronchitis, cough, fever and other respiratory disorders. The present work was carried out to evaluate anti-mycobacterial activity of the ethanolic extract of rhizomes of H. coronarium against respiratory pathogen Mycobacterium tuberculosis (Mtb) with its safety assessment and action on morphology of the bacilli. Anti-mycobacterial activity was assessed using the in-vitro resazurin microtiter plate assay (REMA). The plant extract was further evaluated for its cytotoxicity against human THP-1 macrophages. Ex-vivo intracellular anti-TB activity was also determined using Mtb infected macrophage culture model. Scanning electron microscopy (SEM) was used to examine the morphological changes in Mtb in the presence of plant extract.H. coronarium exhibited promising antimycobacterial activity against drug sensitive Mtb H37Rv and multi drug resistant (MDR) clinical isolates of Mtb with minimum inhibitory concentration (MIC) values ranging from 15.62 to 31.25 µg mL−1. The IC50 (50% inhibitory concentration) of plant extract on THP-1 was found at higher concentration than MIC values against Mtb, indicating that the mammalian cells were not adversely affected at concentrations which are effective against Mtb. The plant extract displayed intracellular killing of bacilli inside the infected macrophages. H. coronarium also produced morphological alteration of the bacilli after 48 and 72 h of treatment. In this study, we conclude that H. coronarium has promising in-vitro and ex-vivo intracellular activity against Mtb and it also displayed activity against rifampicin and isoniazid resistant clinical isolates of Mtb without affecting eukaryotic cells.

Comparison of Conventional Extraction Techniques with Superheated Steam Distillation on Chemical Characterization and Biological Activities of Syzygium aromaticum L. Essential Oil

Syzygium aromaticum L. is an aromatic plant with a significant amount of essential oil (EO), which is used in food, medicine, for flavoring, and in the fragrance industry. The purpose of this study was to comparatively evaluate the chemical composition, yield, and antioxidant and antifungal activities of Syzygium aromaticum essential oils extracted by the conventional hydro-distillation, steam distillation, and the emerging superheated steam distillation methods. It was noticed that the extraction methods significantly influenced the yield, chemical composition, and antioxidant and antimicrobial activities of essential oils. The maximum yield was obtained using superheated steam distillation, followed by hydro-distillation and steam distillation. The antioxidant potential of EO extracts was evaluated following the scavenging of 2,2-dipenyl-1-picrylhydrazyl radicals, hydrogen peroxide scavenging activity and ferric reducing power assays. Results revealed that EO extracted superheated steam distillation exhibited the highest antioxidant activity. GC-MS analysis depicted eugenol (47.94–26.50%) and caryophyllene (20.24–9.25%) as the major compounds of Syzygium aromaticum EOs. The antimicrobial activity of EO extracts was evaluated, via the resazurin microtiter plate assay, microdilution broth assay, and disc diffusion methods, against normal and food pathogenic bacterial and fungal strains. After comparative evaluation, it was observed that superheated steam extracted EO exhibited the highest antimicrobial potential. Overall, methodical evaluation disclosed that superheated steam distillation is an effective method to extract EOs from plant sources, with greater yield and promising biological activities.

In vitro antibacterial activity of andrographolide and hordenine against methicillin-resistant Staphylococcus aureus and Staphylococcus aureus

Bacterial infections are difficult to treat, especially those infected with resistant bacteria. Due to the haphazard use of antibiotics in humans and animals, methicillin-resistant Staphylococcus aureus (MRSA) has developed resistance to practically all commercially available antibacterial drugs. Exploring natural plant compounds with antibacterial activity could be beneficial in the treatment of infections with MRSA and Staphylococcus aureus. Andrographolide, a labdane diterpenoid derived from Andrographis paniculata, and Hordenine, a phenethylamine alkaloid found in Hordeum vulgare, are reported to have antibacterial activity. In the present study, the antibacterial activity of andrographolide and hordenine was tested against MRSA and S. aureus using the Kirby- Bauer agar disc diffusion method and the modified resazurin microtiter plate assay. The results of the study revealed that both andrographolide and hordenine at various concentrations did not show any zone of inhibition against MRSA and S. aureus. Hordenine showed an MIC of 1000 μg/mL against MRSA and S. aureus, but andrographolide had no effect on both MRSA and S. aureus.

GC-MS Analysis and Antimicrobial Assessment of Syzygium cumini (L.) Skeels Seed Ethanol Extract

People use plants to treat infections, and this has led to search of antimicrobials from medicinal plants. In this work, we evaluated the ethanol extract of Syzygium cumini seeds for their antibacterial and antifungal activities. Extraction was performed by maceration method using ethanol. The antimicrobial efficacy of the extract was assessed by agar well diffusion method against ten bacterial species, Bacillus cereus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhi, Serratia marcescens, Staphylococcus aureus and Streptococcus mutans, and five fungal species, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Candida albicans and Mucor sp. Minimum inhibitory concentrations (MICs) of the extract were determined by resazurin microtiter plate assay. Phytochemicals in the extract was identified by gas chromatography mass spectrometry (GC-MS) information. In agar well diffusion method, Gram-negative bacteria such as P. aeruginosa and S. marcescens, Gram-positive bacteria such as B. subtilis and E. faecalis and fungi A. fumigatus were more susceptible showing larger zones of inhibition. In resazurin method, low MICs were recorded for bacteria, B. cereus (<7.8 µg) and P. aeruginosa (15.6 µg) and fungi, A. fumigatus (31.2 µg). Fifteen compounds were identified by GC-MS profiling of the extract. The antimicrobial activity of the extract can be rightly related to the secondary metabolites in the ethanol extract of Syzygium cumini seeds.

Brassica nigra essential oil: In-vitro and in-silico antibacterial efficacy against plant pathogenic and nitrifying bacteria

The present study was aimed to examine the antibacterial potential of Brassica nigra essential oil (BNEO) against Ralstonia solanacearum, causal agent of bacterial wilt and Nitrosomonas sp., the nitrifying bacteria. In poisoned food assay, BNEO showed 100% growth inhibition of R. solancearum at ≥ 125 µg mL−1. Revalidation of findings by volatile assay employing inverted Petri plate technique exhibited 100% bacterial growth inhibition caused by vapors of BNEO, even at 50 µg mL−1 concentration. In the broth microdilution assay, the BNEO exhibited significant antibacterial activity only at higher concentrations (>500 µg mL−1). At 500 µg mL−1, BNEO showed 80% bacterial growth inhibition over control, which was at par with that of streptomycin (5 µg mL−1). In resazurin microtitre-plate assay, the maximum concentration of BNEO, at which color change occurred was 512 µg mL−1 (T9), and thus 512 µg mL−1 was concluded as the minimum inhibitory concentration (MIC). BNEO effectively inhibited the activity of Nitrosomonas spp. with 30-65% nitrification inhibition at the dose of 400 mkg−1 of Urea-N. Homology modeled protein targets assisted computational tool-based novel analysis helped to understand that the antibacterial potency of BNEO is due to preferable binding efficiency of allyl isothiocyanate (AITC), the major active ingredient of BNEO.

Antimycobacterial activity of acetone extract and isolated metabolites from folklore medicinal lichen Usnea laevis Nyl. against drug-sensitive and multidrug-resistant tuberculosis strains

Ethnopharmacological relevanceTuberculosis (Tb) is one of the most infectious diseases caused by Mycobacterium tuberculosis (M.t) with almost 2 million deaths yearly. Although many Tb control programs have been organised, there is an elevated number of Tb cases due to the appearance of extremely drug-resistant and multidrug-resistant (MDR) Tb strains. In the cultures of Venezuelan Andes, fruticose lichen Usnea laevis Nyl. (Usneaceae) with folklore name ‘Barba de Piedra, Tusinya’ is used as a natural remedy for Tb. Aim of the studyThis study was performed to provide a scientific rationale for the folklore usage of U. laevis in treating Tb by validating its antimycobacterial activity against two drug-sensitive and four MDR-Tb strains. Materials and methodsThe mycobacterial inhibitory activities of acetone extract (Ul), fractions (F1-10), and isolated metabolites (1–4) of U. laevis were evaluated against M.t H37Ra using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction menadione assay (XRMA). Furthermore, Ul and 1–4 were subjected to antimycobacterial activity against M.t H37Ra, Mycobacterium smegmatis, and four MDR-Tb (MDR-A8, MDR-V791, MDR-R and MDR-40) strains using resazurin microtitre plate assay (REMA) and cytotoxicity against THP-1 macrophages using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their selectivity index values were also calculated. ResultsInitially, Ul has shown prominent inhibitory activity (IC50 value: 5.44 ± 0.36 μg/ml) and four of its fractions (F1, F2, F5 and F7) also exhibited the best inhibitory activity (IC50 values ranged from 7.46 ± 0.19 to 71.38 ± 2.57 μg/ml) against M.t H37Ra using XRMA. Purification of these bioactive fractions identified four metabolites, namely usnic acid (1), atranorin (2), salazinic acid (3), and lobaric acid (4). From the MIC values of REMA, it was identified that Ul, 1 and 4 were more effective in inhibiting the growth of all four MDR-Tb strains, compared to first-line drug rifampicin. Interestingly, Ul has shown better antimycobacterial activity than 1–4 and rifampicin against MDR-Tb strains may be due to the synergistic effect of its metabolites. Also, the IC50 values of Ul and 1–4 on THP-1 macrophages were found to be far higher than MIC values against tested Tb strains, indicating that THP-1 macrophages were not harmfully affected at concentrations that were effective against Tb strains. Further, the calculated selectivity index values revealed the more active and non-toxicity of Ul, 1 and 4 against MDR-Tb strains than rifampicin. ConclusionsThe current study lends the first evidence for the presence of antimycobacterial metabolites in U. laevis. The results exposed the Andean folklore use of U. laevis for treating Tb, and the key biomarker metabolites were found to be 1 and 4. Hence, it can be concluded that U. laevis can be used as a potential source for the novel drug development for MDR-Tb.

Antimicrobial activity of synthetic antimicrobial peptides loaded in poly-Ɛ-caprolactone nanoparticles against mycobacteria and their functional synergy with rifampicin

Tuberculosis (TB) treatment has become a challenge because of the natural presence of multilayered cell wall rich in lipids which restrict antibiotic permeability within the bacteria. The development of mutations conferring resistance has aggravated the situation. Consequently, maximum pharmaceutical efforts are required to improve the treatment, and antimicrobial peptides (AMPs) with antimycobacterial activity can be exploited as a new treatment strategy against TB. The synergistic interaction between conventional antibiotics and AMPs has broadened its application landscape. To overcome peptide instability and bioavailability issues, encapsulation of these bioactive in biocompatible polymers was adopted. In this study, the effect of synthetic AMPs HHC-8 [KIWWWWRKR] and MM-10 [MLLKKLLKKM] encapsulated in poly (ε-caprolactone) nanoparticles (PCL-NPs) was evaluated against mycobacteria using REMA (Resazurin Microtiter Assay Plate) technique. PCL encapsulation allowed us to load the required amount of peptides, i.e. HHC-8 and MM-10, with an efficiency of ∼ 18.9 ± 5.24 and ∼ 21.1 ± 6.19 % respectively, and sphere size was around 376.5 ± 14.9 nm and 289.87 ± 17.98 nm for PCL-HHC-8-NPs and PCL-MM-10-NPs, respectively. Minimal degradation and sustained release of peptides from nanoparticles improved antimicrobial activity, decreasing the MIC50 from 75 µg/ml to 18.75 µg/ml against M. smegmatis and from 75 µg/ml to 9 µg/ml against M. tuberculosis, respectively. The combinatorial MIC assays of encapsulated AMP with rifampicin antibiotics against M. smegmatis showed synergism between AMP-PCL-NPs and antibiotics with fractional inhibitory concentrations (FICs) around ∼ 0.09. The combinations of AMP NPs also demonstrated synergy against the mycobacteria. Our findings suggest that enhanced efficacy is due to protection offered by AMPs encapsulation resulting in augmentation of membrane permeation by AMPs and enhanced accumulation of antibiotics within mycobacteria resulting in synergy. The study findings might assist in the preclinical development of AMP for the fight against TB.

Synthesis, Characterization and Antimycobacterial Activity of Phenanthrenequinone Thiosemicarbazones and their Ruthenium and Zinc Complexes

The substituted phenanthrenequinone thiosemicarbazones (HL1, HL2 and HL3), and their metal complexes (M = Ru(II) or Zn(II); SM1, SM2, SM3 and SM4) were synthesized and characterized by FT-IR, 1H & 13C NMR and ESI-MS. The spectral data IR, 1H & 13C NMR and ESI-MS indicates two tridentate ligands coordinating in the form of an octahedral geometry. The gas phase geometry of all the zinc(II) and ruthenium(II) complexes was carried by using density functional theory (DFT). The antimycobacterial susceptibility testing at 100 μM of the complexes using resazurin microtiter plate assay resulted in growth inhibition. Complexes SM2, SM3 and SM4 showed good antimycobacterial activity at 100 μM concentration by using rifampicin as inhibition control.

Antimycobacterial Activities of Ruthenium(II) Complexes of Hydroxyl Flavone Hydrazones

The Ru(II) complexes of hydrazine derivatives of different 3-hydroxy 4-substituted flavones was synthesized to assess their biological activity. The ligand 2, 4 dinitrophenyl hydrazones of 3-hydroxy 4´-substituted flavones [where 4´-substituents are -OCH3(HL1), -NO2(HL2), NMe2(HL3), Cl (HL4), OCH2Ph (HL5)] were successfully coordinated with Ru(phen)2Cl2 which consists of the formula, [Ru(phen)2(HL1)]Cl2.2.5H2O (M1R), [Ru(phen)2(HL2)]Cl2(M2R), [Ru(phen)2(HL3)]Cl2(M3R), [Ru(phen)2(HL4)]Cl2.1.5H2O (M4R), [Ru(phen)2(HL5)]Cl2.1.5H2O (M5R). All the synthesized complexes were characterized by elemental analysis, IR, 1H-NMR, UV-Vis, and ESI-MS spectroscopic techniques. The geometry optimization of all complexes was carried by using Gaussian-09. All compounds were studied for antimycobacterial activity using Resazurin microtiter plate assay (REMA) followed by colony-forming unit (CFU) count. The metal complexes showed promising activity against M. smegmatis mc2. The DNA interaction of the complexes was also studied. These studies suggest that hydrazine derivatives of 3-hydroxy 4-substituted flavones and their Ru(II) complexes can be good candidates for antimycobacterial drug development studies.

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-resistantclinical 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 62.5μg/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.

Carprofen elicits pleiotropic mechanisms of bactericidal action with the potential to reverse antimicrobial drug resistance in tuberculosis.

BackgroundThe rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has elevated TB to a major global health priority. Repurposing drugs developed or used for other conditions has gained special attention in the current scenario of accelerated drug development for several global infectious diseases. In a similar effort, previous studies revealed that carprofen, a non-steroidal anti-inflammatory drug, selectively inhibited the growth of replicating, non-replicating and MDR clinical isolates of M. tuberculosis.ObjectivesWe aimed to reveal the whole-cell phenotypic and transcriptomic effects of carprofen in mycobacteria.MethodsIntegrative molecular and microbiological approaches such as resazurin microtitre plate assay, high-throughput spot-culture growth inhibition assay, whole-cell efflux inhibition, biofilm inhibition and microarray analyses were performed. Analogues of carprofen were also synthesized and assessed for their antimycobacterial activity.ResultsCarprofen was found to be a bactericidal drug that inhibited mycobacterial drug efflux mechanisms. It also restricted mycobacterial biofilm growth. Transcriptome profiling revealed that carprofen likely acts by targeting respiration through the disruption of membrane potential. The pleiotropic nature of carprofen’s anti-TB action may explain why spontaneous drug-resistant mutants could not be isolated in practice.ConclusionsThis immunomodulatory drug and its chemical analogues have the potential to reverse TB antimicrobial drug resistance, offering a swift path to clinical trials of novel TB drug combinations.

Identification of Antimycobacterial Agent Using In Silico Virtual Screening, ADME Prediction, Docking, and Molecular Dynamics Simulations Approach.

The widespread hazardous issue of antibiotics resistance can be overcome by the development of target based potent antibacterial agents. Filamentous temperature-sensitive mutant Z (FtsZ), a simpler structural prokaryotic homolog of eukaryotic cytoskeletal tubulin, was considered as a competent target in antibacterial drug discovery. The purpose of the present work is to evaluate the antitubercular activity of virtual hits by funnel-shaped filtering with glide docking, followed by MM-GBSA binding energy and molecular dynamics simulation. Pharmacokinetics and biochemical activity of the computationally screened virtual hits have been studied to focus their potential to inhibit the bacterial cell division. The docking study was performed against the crystal structure of Staphylococcus aureus and Mycobacterium tuberculosis FtsZ protein with the hits obtained from High Throughput Virtual Screening using the Glide module in Schrodinger. ADME profile and 50 ns molecular dynamics simulation studies were performed using the Schrödinger suite. The minimal inhibitory concentration of the test compounds was determined by the colorimetric method by the Resazurin Microtiter plate Assay. The binding of hit molecules T5427054 and 6M356S was mainly supported by van der Waals interaction and an electrostatic component of solvation energy computed by the MM-GBSA method. 50 ns MD simulation built stability and dynamic property of the best-docked complex T5427054/2Q1Y. Both the hit molecules displayed antimycobacterial activity with minimal inhibitory concentration 500 μg/mL. In this study, it is found that new screened hit molecules with better theoretical results could be preferred to use as antimycobacterial agents, and further their structural modification might be improved antimycobacterial properties of hit molecules.

Chemical Composition and Bioactivities of Essential Oil from Leaves of Syzygium cumini (L.) Skeels Native to Punjab, Pakistan.

Herbal medicines are widely used for the treatment of different types of diseases like skin and throat infections and other diseases in developing countries. Syzygium cumini (L.) Skeels fruit, leaves and bark were used for the remedies of different diseases anciently. The aim of the present study was to evaluate the chemical profile of Syzygium cumini leaves essential oil (EO) from Punjab, Pakistan. The essential oil was isolated using hydrodistillation technique and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). Free radical scavenging capacity and antioxidant activity were assessed by using DPPH radical scavenging ability, inhibition of linoleic acid peroxidation, bleaching of β-carotene in linoleic acid system and reducing power assays. Antimicrobial potential was assessed by disc diffusion assay and measurement of minimum inhibitory concentration (MIC) using resazurin microtiter-plate assay. The anti-heme biocrystallization activity of EO was also assessed. The major components (>3%) found in Syzygium cumini leaves EO were β-farnesene (3.42 %), caryophyllenol (3.46 %), terpinen-4-ol (3.61 %), β-myrcene (3.90 %), γ-cadinene (4.09 %), fenchol (4.22 %), cis-β-ocimene (4.40 %) and 5-methyl-1,3,6-heptatriene (4.90 %). Excellent antioxidant, antimicrobial and weak antimalarial potential was observed. It can be concluded that Syzygium cumini leaves EO has potential application for food and pharmaceutical industries.

Isoniazid and verapamil modulatory activity and efflux pump gene expression in Mycobacterium tuberculosis

INTRODUCTION: Resistance to first-line anti-tuberculosis drugs is a major concern in the treatment of the disease. New strategies, such as the use of efflux pump inhibitors (EPIs), are being investigated to improve the outcome of the treatment. Verapamil (VP), one such inhibitor, was shown to inhibit several efflux pump (EP) Mycobacterium tuberculosis proteins and demonstrate synergic activity with anti-TB drugs.OBJECTIVE: To evaluate the combinatory effect of isoniazid (INH) and VP in M. tuberculosis.METHODS: Minimal inhibitory concentrations and combinatory effects of INH+VP were determined using respectively resazurin microtitre assay plate (REMA) and resazurin drugs combination microtitre assay (REDCA). From the results, we selected three bacilli with different susceptibility profiles and assessed their expression of 10 EP genes through quantitative reverse transcription polymerase chain reaction after exposure to INH, VP and INH + VP for 48 h.RESULTS: A significant reduction of INH MIC was observed in INH-susceptible isolates upon combination with VP. In brief, gene expression assays revealed expression patterns that could be correlated with each resistance profile, presence or absence of gene mutations and combinatory effect with VP.CONCLUSION: Combining VP with INH showed important results in drug-susceptible strains, and clinical trials on combined VP + anti-TB drugs should be discussed.

LipF increases rifampicin and streptomycin sensitivity in a Mycobacterium tuberculosis surrogate

BackgroundMortality due to tuberculosis (TB) has increased due to the development of drug resistance, the mechanisms of which have not been fully elucidated. Our research group identified a low expression of lipF gene in Mycobacterium tuberculosis clinical isolates with drug resistance. The aim of this work was to evaluate the effect of lipase F (LipF) expression on mycobacterial drug resistance.ResultsThe effects of expressing lipF from Mycobacterium tuberculosis in Mycobacterium smegmatis on resistance to antituberculosis drugs were determined with resazurin microtiter assay plate and growth kinetics. Functionality of ectopic LipF was confirmed. LipF expression reduced the rifampicin (RIF) and streptomycin (STR) minimum inhibitory concentration (MIC) from 3.12 μg/mL to 1.6 μg/mL and 0.25 μg/mL to 0.06 μg/mL respectively, moreover a reduced M. smegmatis growth in presence of RIF and STR compared with that of a control strain without LipF expression (p < 0.05 and p < 0.01) was shown.ConclusionsLipF expression was associated with increased RIF and STR sensitivity in mycobacteria. Reduced LipF expression may contribute to the development of RIF and STR resistance in Mycobacterium species. Our findings provide information pertinent to understanding mycobacterial drug resistance mechanisms.

Inhibitory activity of traditional plants against Mycobacterium smegmatis and their action on Filamenting temperature sensitive mutant Z (FtsZ)-A cell division protein.

The study was designed to assess whether plant extracts / phytochemical (D-Pinitol) synergistically combine with antituberculosis drugs and act on Mycobacterium smegmatis (M. smegmatis) as well as assess their mode of action on Mycobacterium tuberculosis (M.tb) Filamenting temperature sensitive mutant Z (FtsZ) protein. Resazurin microtitre plate assay (Checker board) was performed to analyze the activity of plant extracts against M. smegmatis. Synergistic behaviour of plant extracts / D-Pinitol with Isoniazid (INH) and Rifampicin (RIF) were determined by time-kill and checker board assays. Elongation of M. smegmatis cells due to this treatment was determined by light microscopy. The effect of Hexane methanol extract (HXM) plant extracts on cell viability was determined using PI/SYTO9 dual dye reporter Live/Dead assay. Action of HXM plant extracts / D-Pinitol on inhibition of FtsZ protein was done using Guanosine triphosphatase (GTPase) light scattering assay and quantitative Polymerase Chain Reaction (qPCR). The Hexane-methanolic plant extract of Acacia nilotica, Aegle marmelos and Glycyrrhiza glabra showed antimycobacterial activity at 1.56 ± 0.03, 1.32 ± 0.02 and 1.25 ± 0.03 mg/mL respectively and that of INH and RIF were 4.00 ± 0.06 μg/mL and 2.00 ± 0.04 μg/mL respectively. These plant extracts and major phytochemical exudate D-Pinitol was found to act synergistically with antimycobacterial drugs INH and RIF with an FIC index ~ 0.20. Time-Kill kinetics studies indicate that, these plant extracts were bacteriostatic in nature. D-Pinitol in conjunction with INH and RIF exhibited a 2 Log reduction in the growth of viable cells compared to untreated. Attempt to elucidate their mode of action through phenotypic analysis indicated that these plant extracts and D-Pinitol was found to interfere in cell division there by leading to an abnormal elongated cellular morphology. HXM extracts and D-Pinitol synergistically combined with the first line tuberculosis drugs, INH and RIF, to act on M. smegmatis. The increase in the length of M. smegmatis cells on treatment with D-Pinitol and HXM extract of the plants indicated that they hinder the cell division mechanism thereby leading to a filamentous phenotype, and finally leading to cell death. In addition, the integrity of the bacterial cell membrane is also altered causing cell death. Further gene expression analysis showed that these plant extracts and D-Pinitol hampers with function of FtsZ protein which was confirmed through in vitro inhibition of FtsZ-GTPase enzymatic activity.

Determination of minimum bactericidal concentration, in single or combination drugs, against Mycobacterium tuberculosis.

Aim: To evaluate an assay to detect minimum bactericidal concentration (MBC) in Mycobacterium tuberculosis, using as single model rifampicin, isoniazid, levofloxacin (LVX) and linezolid (LNZ) and in combination. Material &methods: MBCs were carried out directly from resazurin microtiter assay plate and 3D checkerboard in M. tuberculosis H37Rv and five resistant clinical isolates. Results: The proposed MBC assay showed similar values to those determined by MGIT™, used as control. LVX and LNZ's MBC values were close to their MIC values. LNZ or LVX combined with isoniazid and rifampicin showed MBC value reduced in 63.7% of the assays. Conclusion: The proposed assay to determine MBCs of drugs can be applied to the study of new compounds with anti-M. tuberculosis activity to detect their bactericidal effect and also in laboratory routine for clinical dose adjustment of drugs according to the patient's profile.

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.

Resazurin microtitre plate assay and Sensititre® MycoTB for detection of Mycobacterium tuberculosis resistance in a high tuberculosis resistance setting.

BackgroundRapid diagnosis of drug-resistant Mycobacterium tuberculosis is a challenge in low-income countries. Phenotypic drug susceptibility testing using Sensititre® MycoTB assay and the resazurin microtitre plate assay (REMA) are relatively new innovative methods to determine drug susceptibility.ObjectivesThis study aimed to determine the performance of the Sensititre and REMA for M. tuberculosis drug susceptibility testing in a high-volume tuberculosis reference laboratory.MethodsA laboratory-based study was performed at the Inkosi Albert Luthuli Central Hospital Tuberculosis Laboratory from January 2014 to June 2015. The Sensititre® MycoTB plate and REMA were compared to the gold standard agar proportion method (APM) using 134 stored isolates.ResultsAgreement between the Sensititre® MycoTB plate and APM was observed with 98% sensitivity, 82% specificity, 94% positive and 93% negative predictive values of the Sensititre® MycoTB assay for the detection of rifampicin resistance and 97%, 96%, 99% and 88% for isoniazid resistance. Good categorical agreement between the REMA and the APM was observed among isolates with 89% sensitivity, 68% specificity, 89% positive and 68% negative predictive value for the detection of rifampicin resistance and 95%, 96%, 99% and 81% for isoniazid resistance. Results for the second-line drugs showed elevated minimum inhibitory concentrations for multidrug-resistant and extensively drug-resistant tuberculosis isolates.ConclusionThe REMA and Sensititre® MycoTB plate are attractive alternatives to the gold standard APM for the phenotypic detection of M. tuberculosis drug resistance.