Flavonoids as Novel Efflux Pump Inhibitors and Antimicrobials Against Both Environmental and Pathogenic Intracellular Mycobacterial Species.

Therapeutic treatment options for opportunistic non-tuberculous mycobacterial (NTM) infection and/or serious mycobacterial infections such as tuberculosis (TB) and leprosy are limited due to the spread of antimicrobial resistance mechanism. Plant-derived natural compounds as prospective efflux pump inhibitors may present a promising adjunct to conventional chemotherapy by enhancing mycobacterial susceptibility to antibiotics. This study served to evaluate the antimicrobial and resistance-modifying profile of a range of plant-derived flavonoids against the mycobacterial model strains: M. smegmatis, M. aurum, and M. bovis BCG. The minimum inhibitory concentrations (MICs) of the compounds against the mycobacterial strains were determined using both agar dilution and broth dilution assays, while their efflux inhibitory activity was investigated via an ethidium bromide-based fluorometric assay. All compounds were screened for their synergistic effects with ethidium bromide (EtBr) and rifampicin (RIF) against M. smegmatis. Skullcapflavone II (5,2'-dihydroxy-6,7,8,6'-tetramethoxyflavone, 1) exerted potent antimicrobial activity against M. aurum and M. bovis BCG and considerably increased the susceptibility of M. smegmatis to EtBr and RIF. Nobiletin (5,6,7,8,3',4'-hexamethoxyflavone, 2) was determined to be the most potent efflux-inhibitor in M. aurum and M. smegmatis. However, a connection between strong modulatory and putative efflux activity of the compounds could not be observed. Nevertheless, the results highlight two polymethoxyflavones, skullcapflavone II and nobiletin, with potent antimycobacterial and antibiotic resistance modulating activities as valuable adjuvants in anti-mycobacterial therapies.

Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor.

Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5'-methoxyhydnocarpin (5'-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5'-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5'-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5'-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5'-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial.

Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization.

Biopsy and skin-scraping specimens from 130 leprosy cases across the disease spectrum (56 TT/BT/I, 73 BB/BL/LL, and 1 neuritic case) and 50 healthy contacts were studied to assess the application of gene amplification. The nucleic acids from these clinical specimens were extracted by an integrated freeze-thawing--optimized lysozyme-/proteinase-k treatment-purification and fractionation procedure. The nucleic acids from cultured organisms were isolated by the stepwise procedure earlier standardized at this laboratory. Gene amplification for a 360-bp fragment of the 18-kDa protein gene was carried out using primer and the procedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain reaction product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in specimens with demonstrable acid-fast bacilli. A positivity of 73% in TT/BT/I specimens and 93% in BB/BL/LL specimens was observed. Four combinations were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B+ (3%) and EB-, B- (12%). By combining the blot hybridization with EB staining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by the absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organisms, grown from these specimens. It is recommended that for optimum sensitivity and specificity both EB staining and blot hybridization should be done.

Comparative assessment of viability of M.leprae by mouse foot-pad and fluorescent staining techniques.

Morphological characteristics have been used as a parameter to assess the viability of M.leprae in leprosy patients. However, with the advent of the mouse foot-pad technique, viability of M.leprae is determined by growing the bacilli in the mouse foot-pad. In recent years, a fluorescent staining technique using fluorescent diacetate-ethidium bromide (FDA-EB) has been used to assess the viability of cultivable mycobacteria as well as M.leprae. The purpose of this study was to compare the viability of M.leprae by both mouse foot-pad and fluorescent staining techniques. M.leprae strains from both untreated and treated patients as well as mouse passaged strains of M.leprae were used for the comparison. Percentage of green-stained bacilli in the inoculum was compared with that of multiplication of M.leprae in the mouse foot-pad. It was observed that there was no correlation between the estimates of viable M.leprae by fluorescent staining and by mouse foot-pad inoculation. FDA-EB staining appears to reflect only trends as absence of green staining cells had overall general correlation with loss of infectivity to mouse foot-pad but, the converse was not found to be true.

Effects of activated macrophages on Mycobacterium leprae.

Five alternative methods were used to explore in vitro the effects of normal and activated murine macrophages on the metabolic well-being of intracellular Mycobacterium leprae: fluorescein diacetate-ethidium bromide staining, ATP content, synthesis of phenolic glycolipid 1, and two techniques to quantitate oxidation of palmitic acid. In relatively short-term experiments (7 to 10 days), each of these procedures provided strong evidence that activated macrophages exerted a deleterious effect on the leprosy bacillus. These findings appear to confirm the contention that activated macrophages underlie host resistance to clinical leprosy and limitation of M. leprae growth in paucibacillary leprosy.

Fluorescein diacetate and ethidium bromide staining to determine the viability of Mycobacterium smegmatis and Escherichia coli.

The ability of the fluorescein diacetate and ethidium bromide fluorescent staining method to assess the percentage of viable bacterial cells in suspension was compared with the plate counting method. Mycobacterium smegmatis and Escherichia coli bacterial cell suspensions were incubated at 60 degrees C. At different time intervals samples were taken and the percentage of viable cells in each sample was assessed by the fluorescent staining method and compared with the plate counting method. The fluorescent staining method showed a positive correlation with the plate counting method. However, the viable counts by the plate counting method were lower than the staining method when incubated at 60 degrees C, indicating a lag period in the decay of enzymes after bacterial death. Hence, the fluorescent staining technique can be used to assess the trend of bacterial death rather than to assess to exact number of viable bacilli.

Significance of neither green nor red NGR bacilli in FDA-EB staining.

This Study deals with determination of viability by FDA-EB method. It has been observed that some of the bacilli do not take any colour in FDA-EB preparations. These can be called "neither green nor red" (NGR) bacilli. These non-staining bacilli should be taken into account when reporting viability by FDA-EB method.

Assessing the viability of Mycobacterium leprae by the fluorescein diacetate/ethidium bromide staining technique.

In the present study we have evaluated the Fluorescein Diacetate/Ethidium Bromide (FDA/EB) staining technique to assess the viability of Mycobacterium leprae obtained from biopsies of leprosy patients under different periods of treatment. Bacillary suspensions were obtained from skin punch biopsies and stained with the FDA/EB solution. The average percentage of green cells seen which were deemed to be viable were: 67.2% of green cells in patients without previous treatment; 45.6% in patients with 1 to 6 months of treatment; 25.9% for patients with 7 to 12 months of treatment and 10.5% in patients with 13 to 24 months of treatment. All the patients studied were on multidrug therapy. The differences obtained in the percentages of green cells in the different groups of patients were statistically significant as determined by the Wilcoxon's test. The decrease in the percentage of green cells observed with increasing periods of treatment suggests that the FDA/EB technique correlates with the actual viability of M. leprae. The application of this technique in the routine procedures performed with Hansen's disease patients could be very useful for monitoring the effectiveness of treatment in leprosy patients.

Susceptibilities of Mycobacterium leprae and M. avium complex to the H2O2-Fe-mediated halogenation system supplemented with antimicrobial agents.

The susceptibilities of Mycobacterium leprae and M. avium complex (MAC) to the H2-O2-Fe-mediated halogenation system supplemented with antimicrobial agents were evaluated by fluorescein diacetate-ethidium bromide (FDA/EB) staining. In the case of M. leprae, the number of greenstained bacteria (intact cells) was reduced in the presence of the H2O2-Fe-mediated halogenation system supplemented with agents possessing antileprosy activity, such as rifampin, 4,4'-diaminodiphenylsulfone (dapsone), clofazimine, and ofloxacin. In the case of the MAC strain, although viable units of the organisms were reduced by the halogenation system alone, the number of greenstained cells in the FDA/EB stain was not reduced, even when the halogenation system was used in combination with ofloxacin. Because stainability of the cells is related to structural and functional intactness of the membrane, differences between M. leprae and the MAC strain imply possible differences in the rigidity of the cell membrane.

Effect of chemotherapy on viability of Mycobacterium leprae as determined by ATP content, morphological index and FDA-EB fluorescent staining.

Viable bacterial populations were estimated in bacilli purified from 105 biopsies from 40 untreated and 65 multibacillary leprosy patients treated with multidrug therapy (MDT) for varying periods. The bacilli were purified and viability was determined by ATP content, morphological index (MI), and fluorescein diacetate-ethidium bromide (FDA-EB) staining. Viable populations were calculated, taking 3.58 x 10(-15) g/solid bacillus as the mean ATP content of a viable unit of Mycobacterium leprae. The proportion of viable bacilli was also estimated in the same specimens using solid-staining (MI) and green-staining bacilli by the FDA-EB method. In the untreated cases, the positive viability by ATP assay was 100%, 92% by MI, and 100% by FDA-EB. ATP content per solid bacillus was relatively constant, which was not the case with ATP content per green-staining bacillus. While the MI was zero in all cases, viable bacilli could still be detected by ATP estimations in 5 of the 32 (16%) patients after 2 years of MDT and in 1 of the 20 (5%) patients after 3 years of MDT. No viable bacilli could be detected even by this method beyond 3 years of MDT. On the other hand, green-staining bacilli were demonstrable in 7/32 (22%) of cases after 2 years of MDT, 2/20 (10%) after 3 years of MDT, and 1/13 (8%) after more than 3 years of treatment, indicating that the FDA-EB staining and ATP assay did not detect the same populations. A determination of the ATP content of M. leprae could be used as a reliable and sensitive tool for determining viability of the bacilli.

Comparison of radiometric macrophage assay and fluorescein diacetate/ethidium bromide staining for evaluation of M. leprae viability.

Earlier studies from our laboratory reported that a radiometric Mycobacterium leprae resident macrophage assay was a useful in vitro indicator of bacillary viability with good correlation with the established mouse foot pad model. The present study compares our assay with the recently described fluorescein diacetate/ethidium bromide (FDA/EB) method. M. leprae extracted from the dermal lesions of 73 bacilliferous leprosy patients were tested concurrently by both techniques. Good correlation (r = 0.52, p less than 0.001) was found between the radiometric assay evaluating DNA synthesis and the FDA/EB staining reflecting the presence of active esterase enzyme. In addition, the utility of the FDA/EB staining in the monitoring of therapy was established. Twenty-two patients treated for greater than 1 year showed lower numbers of green fluorescing bacilli when compared to 19 untreated or short-term-treated individuals.

Viability of Mycobacterium leprae: a comparison of morphological index and fluorescent staining techniques in slit-skin smears and M. leprae suspensions.

In a comparison of the estimation of Mycobacterium leprae viability by morphology and the fluorescent vital dyes FDA/EB and R123/EB, the latter techniques were more satisfactory using suspensions and slit-skin smears of M. leprae bacilli. Both FDA/EB and R123/EB seem to more accurately reflect viability after freeze/thaw cycles and heating, and are able to detect lower percentages of viable bacilli. In addition, the fluorescent vital dye techniques are both simple and less open to subjective interpretation than the conventional estimation of the morphological index.

Staining tissue-derived Mycobacterium leprae with fluorescein diacetate and ethidium bromide.

A fluorescent staining procedure incorporating the use of fluorescein diacetate (FDA) and ethidium bromide (EB) has previously been shown to accurately measure the viability of saprophytic mycobacterial cells. Green-stained cells were shown to be viable and red-stained cells, dead. Staining Mycobacterium leprae cells with FDA/EB, however, was complicated by interfering tissue components which masked the presence of stained bacteria. A petroleum ether separation technique enables M. leprae to be segregated from armadillo liver tissue components and permitted M. leprae to be stained qualitatively equal to the saprophytic mycobacteria. An alternative and technically simpler method of staining M. leprae from human skin biopsies and mouse foot pads was developed which permitted the initiation of a clinical assessment of the staining method. Preliminary data indicate that patients who have undergone three or 24 months of chemotherapy possess a significantly lower percentage of green-stained M. leprae in their tissues than untreated patients. This would be expected if the FDA/EB staining method was providing an accurate measure of viability. M. leprae cells obtained from mouse foot pads which were harvested 5-13 months post-infection displayed more than 90% green-stained cells. There was no correlation between the FDA/EB staining method and the morphological index.

Effect of mutation, electric membrane potential, and metabolic inhibitors on the accessibility of nucleic acids to ethidium bromide in Escherichia coli cells.

The uptake of ethidium bromide by Escherichia coli K 12 cells has been studied by using 14C-labeled ethidium and spectrofluorometry on three E. coli strains: the first one (AB1157) has an ethidium-resistant phenotype; the second one derives from the first one after a single mutation (at 10 min on the E. coli genetic map) and has an ethidium-sensitive (Ebs) phenotype; the third one is the acrA strain which appeared to have the same phenotype as the Ebs strain. When the cells are in exponential growth, no ethidium enters wild-type cells, and a very limited amount of ethidium enters Ebs and acrA cells. Massive quantities of ethidium enter AB1157, Ebs, and acrA cells treated by uncouplers and respiring Ebs cells treated by the membrane ATPase-inhibitor dicyclohexylcarbodiimide. A small amount of ethidium enters cells treated in M9 succinate medium by metabolic inhibitors such as KCN or cells starved with oxygen in the same M9 medium. The amount of ethidium and ethidium dimer retained at equilibrium by either type of cell, and by cells infected by T5 phage, as well as the kinetics of influx and efflux, has been measured under a variety of situations (membrane energized or not, and/or membrane ATPase inhibited or not). Furthermore, it was shown that ethidium binds to both RNA and DNA when it enters CCCP-treated wild-type E. coli cells, whereas it binds mainly to DNA when it enters Ebs and acrA cells in exponential growth. As it will be discussed, it is difficult to account for the EthBr uptake by invoking only membrane functions and active transport. Therefore, it is proposed that the variations of the nucleic acid accessibility in E. coli cells might play a role in the control of this uptake. Accordingly, in ethidium-sensitive cells, the mutation would have caused a significant part of the chromosomal DNA (10-20%) to become accessible to ethidium. Hansen [Hansen M. T. (1982) Mutat. Res. 106, 209-216], after a study of the photobinding of psoralen to nucleic acids in the acrA mutant, also suggested that DNA environment was modified in acrA cells.

A fluorescent staining procedure for determining the viability of mycobacterial cells.

A fluorescent staining procedure has been developed which rapidly, accurately, and economically measures the viability of mycobacterial cells. M. smegmatis and M. phlei have served as prototype organisms to establish conditions which ensure optimal staining. The staining method incorporates the use of the fatty acid ester fluorescein diacetate (FDA) and ethidium bromide (EB). Non-polar, non-fluorescent FDA enters live cells where it is enzymatically hydrolyzed by acetylesterase to polar, fluorescent fluorescein which rapidly accumulates in the cytoplasm. These cells appear green when viewed under incident ultraviolet illumination. Ethidium bromide enters dead cells and intercalates between the bases of DNA molecules. These cells appear red-orange under UV illumination. Live cells are, therefore, identified on the basis of possessing acetylesterase and their ability to exclude EB; whereas dead cells are identified on the basis of lacking acetylesterase and their inability to exclude EB. The feasibility of applying the staining procedure of M. leprae has been investigated and the results are encouraging. Our findings reveal that armadillo-derived M. leprae possess acetylesterase and, therefore, stain green. M. leprae cell suspensions exposed to adverse physico-chemical conditions give rise to high proportions of red-stained cells as would be expected if the cells are being killed. An alternative means of determining the viability of M. leprae appears to be feasible.

In-vitro modification of membrane receptors on cells of the mononuclear phagocyte system.

EA and EAC receptors have been studied on non-elicited and paraffin-induced macrophages, under a variety of culture conditions in vitro, for up to 7 days. A large decrease in the number of macrophages showing EAC receptors was found after treatment of the cells with BCG, but not "inert" particles such as latex and zymosan. This was reversible by 7 days. In the presence of a toxic material, Al(OH)3, both EA and EAC receptors were partially lost. The results obtained have been related to previous results obtained with cryostat sections of human leprosy skin lesions.