Optimization of a Rapid Viability Assay for Mycobacterium avium subsp. paratuberculosis by Using alamarBlue

A microtiter alamarBlue assay was adapted and optimized for Mycobacterium avium subsp. paratuberculosis. Using cell concentrations ranging from 10⁴ to 10⁸ CFU/ml, a minimum incubation time to indicate viability was obtained after 24 h. Rifampin (rifampicin) was used to demonstrate that this method has applications for high-throughput screening against M. avium subsp. paratuberculosis.Mycobacterium avium subsp. paratuberculosis is a chronic enteric pathogen which is widely distributed throughout the food chain (4, 16). Its association with Johne''s disease in cattle is economically significant, with the United States alone suffering losses of $1.5 billion a year (6). Furthermore, its potential to cause human disease is disconcerting and controversial (7, 10, 11, 13). Therefore, the identification and development of novel anti-M. avium subsp. paratuberculosis agents are urgently required. In order to facilitate this, it is important to have available rapid anti-M. avium subsp. paratuberculosis assays permitting high-throughput analysis. A microtiter alamarBlue assay (currently untested for M. avium subsp. paratuberculosis) is a reliable means of determining cellular viability in bacteria (9). This rapid and inexpensive assay lends itself to a high-throughput screening format and has been shown to be applicable to some species of mycobacteria (3, 15). Furthermore, its correlation with other more expensive methods for determining mycobacterium viability is high, between 93 and 100% (1, 5, 15). These include the Mycobacteria Growth Indicator Tube, the Bactec radiometric method, and luciferase reporter systems. This study set out to establish and optimize a microtiter alamarBlue assay for a broad range of M. avium subsp. paratuberculosis titers and to evaluate applications for this assay, including high-throughput screening of novel anti-M. avium subsp. paratuberculosis compounds, and antibiotic resistance profiling of M. avium subsp. paratuberculosis.M. avium subsp. paratuberculosis (CIT03) was isolated from the feces of an infected cow, as described by Ristow et al. (12), and cultivated on Herrold''s egg yolk medium agar supplemented with mycobactin J (2 μl/ml), amphotericin B (50 μg/ml), vancomycin (50 μg/ml), and nalidixic acid (50 μg/ml) for 16 weeks at 37°C. The identity of M. avium subsp. paratuberculosis was confirmed using acid-fast staining, mycobactin dependency, and PCR analysis as previously described (2, 8, 14). To generate sufficient biomass, M. avium subsp. paratuberculosis was subsequently grown in Middlebrook 7H9 broth (MB broth), supplemented with oleic acid, albumin, dextrose, catalase (10%; Becton Dickinson), glycerol (0.2%), and mycobactin J (0.2%). This generally took 16 weeks.Prior to the assay, 10 ml of the 16-week culture was centrifuged at 15,000 rpm for 20 min. The pellet was washed in fresh MB broth and resuspended in 10 ml of fresh supplemented MB broth containing 0.2% mycobactin J. The turbidity was adjusted to match McFarland standard no. 1 (3 × 10⁸ CFU/ml). From this suspension, a series of 1:5 dilutions ranging from 3 × 10⁸ to 9.6 × 10⁴ CFU/ml was set up in MB broth (5-ml volumes), using sterile Falcon tubes. The microtiter plate was organized into rows B, C, D, E, F, and G. Two-hundred-microliter aliquots of 3 × 10⁸ CFU/ml M. avium subsp. paratuberculosis were added to 10 wells in row B. Two-hundred-microliter aliquots of 6 × 10⁷ CFU/ml were added to row C, 1.2 × 10⁷ CFU/ml to row D, 2.4 × 10⁶ CFU/ml to row E, 4.8 × 10⁵ CFU/ml to row F, and 9.6 × 10⁴ CFU/ml to row G. This assay was allowed to progress over a period of 11 days. Twenty microliters (10% of the volume in the well) of a fresh alamarBlue reagent (AbD Serotec) was added, with mixing, to each column on each sampling day. Plates were covered and resealed with Parafilm and incubated at 37°C after the addition of the dye. Absorbance readings at 570 and 600 nm were then taken at 6 h, 24 h, and 48 h for each column. The assay was performed in triplicate, and percent reduction values of alamarBlue were determined using the appropriate formula (www.biokom.com.pl/files/alamarblue.pdf).In terms of optimization, this assay examined the influence of cell numbers, the cellular incubation time, and the optimal incubation time with alamarBlue. All three had a significant impact on color development and percent reduction of the dye. For the highest concentration of cells (3 × 10⁸ CFU/ml) (Fig. ​(Fig.1a),1a), a strong reduction of the dye was observed after 1 day of cellular incubation. Further incubation of the cells or incubation with the dye did not result in an appreciable increase in dye reduction values. Indeed, a decrease in the percent reduction was noted, most likely due to buffering agents reaching their maximum buffering efficiencies in the reagent mix (www.abdserotec.com/about/alamarblue). At the mid-range cellular levels (2.4 × 10⁶ CFU/ml) (Fig. ​(Fig.1b),1b), detectable dye reduction occurred after 2 days. The reduction was substantial after day 4. At the lowest concentration of cells (9.6 × 10⁴ CFU/ml) (Fig. ​(Fig.1c),1c), a noticeable change in dye reduction was observed after 9 days. While longer incubation with alamarBlue led to greater dye reduction with a given cell titer, as shown with each suspension at 6, 24, and 48 h, the 24-h reading was considered sufficient to give a clear indication of viability.Open in a separate windowFIG. 1.Optimization of alamarBlue conditions using 3 × 10⁸ CFU/ml (a), 2.4 × 10⁶ CFU/ml (b), and 9.6 × 10⁴ CFU/ml (c) over 11 days. Following the addition of alamarBlue, readings were taken at 6, 24, and 48 h.Percent reduction of the dye was standardized to 10, 20, 40, and 60% for each concentration of cells (Table ​(Table1).1). These values serve as definitive indicators of metabolic activity and may be used for multiple applications, such as comparing the relative viabilities of strains, or the influence of media composition or environmental stress on M. avium subsp. paratuberculosis. In particular, we feel that this assay is suited to comparing the relative efficacies of multiple anti-M. avium subsp. paratuberculosis compounds and/or antibiotic resistance profiling in a high-throughput screening format. The success of this assay requires strict adherence to specific cell numbers, growth phases, and their equivalent incubation times.

TABLE 1.

Required time taken for M. avium subsp. paratuberculosis to reduce alamarBlue

The effect of sourdough and calcium propionate on the microbial shelf-life of salt reduced bread

The consumption of low-salt bread represents an efficient way to improve public health by decreasing cardiovascular health issues related to increased intakes of sodium chloride (NaCl). The reduction of NaCl influences the bread quality characteristics, in particular the shelf-life. Calcium propionate (CP) is commonly used in bread as an antifungal agent. Alternatively, sourdough can be used as a natural preservative. This work addresses the feasibility of NaCl reduction in wheat bread focussing on shelf-life and the compensation using sourdough as well as chemical preservatives. The impact of NaCl reduction and the addition of preservative agents in conjunction with different NaCl concentrations on the shelf-life of bread were tested under 'environmental' conditions in a bakery as well as using challenge tests against selected fungi. The challenge tests were performed using fungi commonly found in the bakery environment such as Penicillium expansum, Fusarium culmorum and Aspergillus niger. NaCl reduction decreased the shelf-life by 1-2?days. The addition of sourdough with antifungal activity prolonged the shelf-life to 12-14?days whereas the addition of 0.3?% calcium propionate prolonged the shelf-life to 10-12?days only. The fungal challenge tests revealed differences in the determined shelf-life between the different fungi based on their resistance. Similar antifungal performance was observed in sourdough breads and calcium propionate breads when tested against the different indicator moulds. The findings of this study indicate that addition of sourdough fermented using a specifically selected antifungal Lactobacillus amylovorus DSM 19280 can replace the chemical preservative calcium propionate addition and compensate for the reduced level and, therefore, guarantee the product safety of low-salt bread.     

Syntheses of 3-carbomethoxy-4-(aryl)piperidines and in vitro and in vivo pharmacological evaluation: identification of inhibitors of the human dopamine transporter

A series of 3-carbomethoxy-4-(aryl-substituted)piperidines with various aryl groups were synthesized and examined for binding and reuptake inhibition at the human dopamine transporter, the human serotonin transporter, and the human norepinephrine transporter. The binding potency and reuptake inhibition efficacy was compared with that of (-)-cocaine to determine the significance of removing the two-carbon bridge of the cocaine nucleus on the inhibition of transporter binding and reuptake. Of the transporters examined, the substituted piperidines were relatively selective for the human dopamine transporter. In all cases examined, the cis-diastereomer of the 3-carbomethoxy-4-(aryl-substituted)piperidine was observed to be a more potent inhibitor of the human dopamine transporter than the trans diastereomer. Based on the K(i) (binding) and IC(50) (reuptake inhibition) values obtained, the most potent inhibitor of the series was cis-3-carbomethoxy-4-(4'-chlorophenyl)piperidine, and this compound suppressed spontaneous- and cocaine-induced stimulation in non-habituated male Swiss-Webster mice. The conclusion is that substantial portions of the cocaine structure can be dissected away to provide compounds with significant binding and reuptake inhibition of the human dopamine transporter.     

Inhibition of serotonin and norepinephrine reuptake and inhibition of phosphodiesterase by multi-target inhibitors as potential agents for depression

Compounds possessing more than one functional activity incorporated into the same molecule may have advantages in treating complex disease states. Balanced serotonin/norepinephrine reuptake inhibitors (SNRIs) (i.e., (R)- and (S)-norduloxetine) were chemically linked to a PDE4 inhibitor via a five carbon bridge. The new dual SNRI/PDE4 inhibitors (i.e., (R)-15 and (S)-15) showed moderately potent serotonin reuptake inhibition (IC₅₀ values of 442 and 404 nM, respectively) but low reuptake inhibition of norepinephrine (IC₅₀ values of 2097 and 2190 nM, respectively) in vitro. The dual SNRI/PDE4 inhibitors (i.e., (R)-15 and (S)-15) also inhibited PDE4D2 (i.e., K_i values of 23 and 45 nM, respectively). Due to their synergistic functional activity, SNRI/PDE4 inhibitors may be effective in treating diseases such as depression.     

Hydropathic analysis of the free energy differences in anthracycline antibiotic binding to DNA

Molecular models of six anthracycline antibiotics and their complexes with 32 distinct DNA octamer sequences were created and analyzed using HINT (Hydropathic INTeractions) to describe binding. The averaged binding scores were then used to calculate the free energies of binding for comparison with experimentally determined values. In parsing our results based on specific functional groups of doxorubicin, our calculations predict a free energy contribution of –3.6 ± 1.1 kcal mol^–1 (experimental –2.5 ± 0.5 kcal mol^–1) from the groove binding daunosamine sugar. The net energetic contribution of removing the hydroxyl at position C9 is –0.7 ± 0.7 kcal mol^–1 (–1.1 ± 0.5 kcal mol^–1). The energetic contribution of the 3′ amino group in the daunosamine sugar (when replaced with a hydroxyl group) is –3.7 ± 1.1 kcal mol^–1 (–0.7 ± 0.5 kcal mol^–1). We propose that this large discrepancy may be due to uncertainty in the exact protonation state of the amine. The energetic contribution of the hydroxyl group at C14 is +0.4 ± 0.6 kcal mol^–1 (–0.9 ± 0.5 kcal mol^–1), largely due to unfavorable hydrophobic interactions between the hydroxyl oxygen and the methylene groups of the phosphate backbone of the DNA. Also, there appears to be considerable conformational uncertainty in this region. This computational procedure calibrates our methodology for future analyses where experimental data are unavailable.     

Bivalent biogenic amine reuptake inhibitors

A series of aryltropane-based bivalent ligands was prepared and investigated for binding potency and for their ability to inhibit reuptake of human dopamine, serotonin and norepinephrine transporters. The bivalent ligand 4, comprised of linking an aryltropane by an octamethylene spacer showed high efficacy for the human dopamine transporter and had a discrimination ratio of 130.     

The effect of conjugated linoleic acid on calcium absorption and bone metabolism and composition in adult ovariectomised rats

The effect of conjugated linoleic acid (CLA) on postmenopausal bone metabolism has not been investigated. Therefore, forty-three adult ovariectomised (OVX) rats (8-9 rats per group) were fed either a control diet containing 40 g/kg soyabean oil (SBO diet) or the SBO diet with 0 (control OVX), 2.5, 5 or 10 g/kg of CLA (replacing soybean oil) for 9 weeks. A group of sham-operated (SH) rats were fed the SBO diet. OVX rats had significantly (P<0.05) lower femoral bone mineral density and macromineral concentration, and intestinal Ca absorption compared to SH rats. CLA supplementation had no effect on these parameters. Ex vivo PGE(2) biosynthesis by bone and urinary Pyr and Dpyr (markers of bone resorption) were significantly higher (P<0.001) in control OVX rats compared with SH rats, and were significantly (P<0.001) lowered by CLA supplementation with 5 and 10, but not 2.5 g/kg diet in OVX rats. In conclusion, CLA supplementation appeared to reduce the rate of bone resorption in adult OVX rats.     

Some distinctions between flavin-containing and cytochrome P450 monooxygenases

This minireview summarizes information concerning the differences and similarities of the human flavin-containing- (FMO, E.C. 1.14.13.8) and the cytochrome P450-monooxygenases (CYP, E.C. 1.14.14.1). Human FMO oxygenates soft nucleophiles. CYP mainly catalyzes C-H abstraction but also oxidizes nitrogen- and sulfur-containing compounds. Both FMO and CYP generally convert lipophilic compounds into more hydrophilic metabolites. The mechanism by which each monooxygenase operates is quite distinct. Sometimes, CYP or FMO bioactivate chemicals to reactive metabolites but to date, drug toxicity thus far observed in the clinic is mainly the result of CYP-dependent oxidation. Both FMO and CYP possess genetic variability that may contribute to inter-individual variability observed for drug metabolism. In contrast to CYP, FMO is not induced or readily inhibited and potential adverse drug-drug interactions are minimized for drugs prominently metabolized by FMO. These properties may provide advantages in drug design, and by incorporating FMO detoxication pathways into drug candidates, more drug-like materials may emerge.     

The effect of conjugated linoleic acid on the viability and metabolism of human osteoblast-like cells

Studies in experimental animals and murine osteoblast cells in culture have produced conflicting findings on the effect of conjugated linoleic acid (CLA) on bone formation. The present study investigated the influence of CLA on viability and metabolism of two human osteoblast-like cell lines (SaOS2 and MG63). Both cell lines were exposed to increasing concentrations (0-50 microM) of CLA either as pure cis (c) 9: trans (t) 11 and t10:c12 CLA isomers or a blend of isomers, or linoleic acid (C18:2). Cell cytotoxicity and degree of DNA fragmentation were unaffected by any fatty acid treatment. PGE2 biosynthesis by both cell lines was variably reduced by CLA isomer blend and t10:c12 CLA, but not c9:t11 CLA. Alkaline phosphatase activity was variably increased by all CLA treatments. These results suggest a lack of cytotoxic effect of CLA on human osteoblast-like cells and tentatively suggest a possible beneficial effect on bone formation in humans.