Screening biofilm eradication activity of ethanol extracts from foodstuffs: potent biofilm eradication activity of glabridin, a major flavonoid from licorice (Glycyrrhiza glabra), alone and in combination with ɛ-poly-L-lysine.

The ethanol extracts of 155 different foodstuffs containing medicinal plants were investigated for their biofilm eradication activities against pathogenic bacteria. A combined method of a colorimetric microbial viability assay based on reduction of a tetrazolium salt (WST-8) and a biofilm formation technique on the 96-pins of a microtiter plate lid was used to screen the biofilm eradication activities of foodstuffs. The ethanol extracts of licorice (Glycyrrhiza glabra) showed potent biofilm eradication activities against Streptococcus mutans, Staphylococcus aureus, and Porphyromonas gingivalis. Among the antimicrobial constituents in licorice, glabridin had the most potent eradication activities against microbial biofilms. The minimum biofilm eradication concentration of glabridin was 25-50 µg/ml. Furthermore, the combination of glabridin with ɛ-poly-L-lysine, a food additive, could result in broad biofilm eradication activities towards a wide variety of bacteria associated with infection, including Escherichia coli and Pseudomonas aeruginosa.

A rapid and simple measurement method for biofilm formation inhibitory activity using 96-pin microtiter plate lids.

The purpose of this study was to develop a rapid and simple measurement method for biofilm formation inhibitory activity, and to screen food additives and foodstuffs that inhibit biofilm formation. The measurement method for biofilm formation inhibitory activity was developed by combining biofilm formation on pins of microtiter plate lids and staining using crystal violet. The optimum conditions for biofilm formation on the pins were established for seven Gram-positive and six Gram-negative bacteria by investigations of media, incubation time, and pin materials. Minimum concentrations of food additives required to inhibit biofilm formation were determined using the proposed method. The values obtained by the proposed and conventional methods agreed well. In addition, by sequential measurements of minimum inhibitory concentrations and minimum bactericidal concentrations using the proposed method, mechanisms of inhibition of biofilm formation were assessed. Furthermore, inhibitory activities of the water extracts of 498 different plant foodstuffs on biofilm formation by Streptococcus mutans were measured; five of the extracts showed potent inhibitory activities. The method proposed here circumvents the tedious and time-consuming conventional method in which biofilms are cultivated on the bottom of wells of microtiter plates.

Biofilm Eradication Activity of Herb and Spice Extracts Alone and in Combination Against Oral and Food-Borne Pathogenic Bacteria.

The purpose of this study was to select herbs and spices with potent biofilm eradication activities. Further, the combined effects of herb and spice extracts against pathogenic biofilms were evaluated. The biofilm eradication activities of ethanol extracts of 104 herbs and spices were measured by combining a colorimetric microbial viability assay with a biofilm formation technique. Ethanol extract of clove had potent biofilm eradication activities against Escherichia coli, Porphyromonas gingivalis, and Streptococcus mutans. Ethanol extracts of eucalyptus and rosemary had potent biofilm eradication activities against P. gingivalis, Staphylococcus aureus and S. mutans. The combination of extracts of clove with eucalyptus or rosemary showed synergistic or additive effects, or both, on biofilm eradication activities. The main biofilm inhibitors in the ethanol extracts of clove, eucalyptus and rosemary were eugenol, macrocarpals and carnosic acid, respectively. The combinations of extracts of clove with eucalyptus or rosemary had potent biofilm eradication activities against oral and food-borne pathogenic bacteria. The findings of the present study reveal that specific combinations of herb and spice extracts may prevent and control biofilm-related oral diseases, food spoilage, and food poisoning.

Fatty Acid Potassium Had Beneficial Bactericidal Effects and Removed Staphylococcus aureus Biofilms while Exhibiting Reduced Cytotoxicity towards Mouse Fibroblasts and Human Keratinocytes.

Wounds frequently become infected or contaminated with bacteria. Potassium oleate (C18:1K), a type of fatty acid potassium, caused >4 log colony-forming unit (CFU)/mL reductions in the numbers of Staphylococcus aureus and Escherichia coli within 10 min and a >2 log CFU/mL reduction in the number of Clostridium difficile within 1 min. C18:1K (proportion removed: 90.3%) was significantly more effective at removing Staphylococcus aureus biofilms than the synthetic surfactant detergents sodium lauryl ether sulfate (SLES) (74.8%, p < 0.01) and sodium lauryl sulfate (SLS) (78.0%, p < 0.05). In the WST (water-soluble tetrazolium) assay, mouse fibroblasts (BALB/3T3 clone A31) in C18:1K (relative viability vs. control: 102.8%) demonstrated a significantly higher viability than those in SLES (30.1%) or SLS (18.1%, p < 0.05). In a lactate dehydrogenase (LDH) leakage assay, C18:1K (relative leakage vs. control: 108.9%) was found to be associated with a significantly lower LDH leakage from mouse fibroblasts than SLES or SLS (720.6% and 523.4%, respectively; p < 0.05). Potassium oleate demonstrated bactericidal effects against various species including Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Clostridium difficile; removed significantly greater amounts of Staphylococcus aureus biofilm material than SLES and SLS; and maintained fibroblast viability; therefore, it might be useful for wound cleaning and peri-wound skin.

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria.

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.

Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate.

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances.

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts.

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.

New tetrazolium method for phosphatase assay using ascorbic acid 2-phosphate as a substrate.

A new method to assay alkaline and acid phosphatases assay using ascorbic acid 2-phosphate (AsA-P) and ditetrazolium salt nitroblue tetrazolium chloride (NBT) was developed. AsA-P is hydrolyzed in the presence of phosphatase to yield ascorbic acid. In turn, the ascorbic acid reduces NBT directly or indirectly, opening the tetrazole ring to produce an insoluble formazan as a colored precipitate. The proposed method for alkaline phosphatase was compared with a conventional method in which 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used in combination with NBT in the dot blots of a dilution series of beta-lactoglobulin. AsA-P reduced NBT more effectively than BCIP in the presence of alkaline phosphatase. AsA-P could be also used as the chromogenic substrate for an acid phosphatase assay in the presence of phenazinium methylsulfate and NBT.

Flow-injection fluorometric quantification of pyruvate using co-immobilized pyruvate decarboxylase and aldehyde dehydrogenase reactor: Application to measurement of acetate, citrate and l-lactate.

A flow-injection system for the quantification of pyruvate based on the coupled reactions of pyruvate decarboxylase (PDC) and aldehyde dehydrogenase (AlDH) was conceived and optimized. A co-immobilized PDC and AlDH reactor was introduced into the flow line. Sample and reagent (NAD(+)) were injected into the flow line by an open sandwich method and the increase of NADH produced by the immobilized-enzyme reactor was monitored fluorometrically at 455nm (excitation at 340nm). Linear relationships between the responses and concentrations of pyruvate were observed in the ranges of 2.0x10(-5) to 1.5x10(-3)M at the flow rate of 1.0mlmin(-1) and 5.0x10(-6) to 1.0x10(-3)M at the flow rate of 0.5mlmin(-1). The relative standard deviation for 10 successive injections was 0.95% at the 1.0mM level. This FIA system for pyruvate was applied to the measurement of acetate, citrate and l-lactate.

Sequential fluorometric quantification of malic acid enantiomers by a single line flow-injection system using immobilized-enzyme reactors.

A method for the sequential enantiomeric quantification of d-malate and l-malate by a single line flow-injection analysis was developed using immobilized-enzyme reactors and fluorescence detection. An immobilized d-malate dehydrogenase (d-MDH) reactor and an immobilized l-malate dehydrogenase (l-MDH) reactor were introduced into the flow line in series. Sample and coenzyme (NAD(+) or NADP(+)) were injected into the flow line by an open sandwich method. d-Malate was selectively oxidized by d-MDH when NAD(+) was injected with a sample. When NADP(+) was injected with a sample, l-malate was oxidized only by l-MDH. NADH or NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455nm (excitation at 340nm). Linear relationships between the responses and concentrations of d-malate and l-malate were observed in the ranges of 1 x 10(-6)-1 x 10(-4)M and 1 x 10(-6)-2 x 10(-4)M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.1mM level. This analytical method was applied to the sequential quantification of d-malate and l-malate in fruit juices and soft drinks, and the results showed good agreement with those obtained using conventional method (F-kit method).

Metabolism of benzoquinone by yeast cells and oxidative characteristics of corresponding hydroquinone: application to highly sensitive measurement of yeast cell density by using benzoquinone and a chemiluminescent probe.

The metabolic efficiency of seven derivatives of 1,4-benzoquinone (BQ) by yeast cells and the oxidative characteristics of the corresponding hydroquinones (HQs) were studied by electrochemical, spectrophotometric and chemiluminescent methods. The spectrophotometric method was based on the reduction of a tetrazolium salt to formazan dye during the autoxidation of HQs generated by yeast cells under alkaline conditions. The amounts of HQs detected directly by the electrochemical method did not agree with those calculated from the formazan dye obtained by the spectrophotometric method. A tetrazolium salt was reduced to a formazan dye by both the superoxide anion radical (O2-*) generated during the autoxidation of 2,3,5,6-tetramethyl-1,4-HQ and by HQ itself. Little formazan dye was formed, and hydrogen peroxide (H2O2) was then finally produced during the autoxidation of 1,4-HQ or 2-methyl-1,4-HQ. Formazan dye and H2O2 were generated at a certain ratio during the autoxidation of derivatives of dimethyl-1,4-HQ or 2,3,5-trimethyl-1,4-HQ. The analytical method based on chemiluminescence with lucigenin and 2,3,5,6-tetramethyl-1,4-BQ was applied to highly sensitive measurement of the yeast cell density. A linear relationship between the chemiluminescence intensity and viable cell density was obtained in the range of 1.2 x 10(3) - 4.8 x 10(4) cells/ml. The detection limit was 4.8 x 10(2) cells/ml.

Spectrophotometric assay of yeast vitality using 2,3,5,6-tetramethyl-1,4-benzoquinone and tetrazolium salts.

A method for the spectrophotometric assay of yeast vitality was developed using 2,3,5,6-tetramethyl-1,4-benzoquinone and tetrazolium salts. The metabolic efficiency of 2,3,5,6-tetramethyl-1,4-benzoquinone by yeast cells was used as an index of yeast vitality. 2,3,5,6-Tetramethyl-1,4-benzoquinone was reduced to 2,3,5,6-tetramethyl-1,4-hydroquinone by yeast cells. Then, the superoxide anion radicals generated from O2 by reduction with 2,3,5,6-tetramethyl-1,4-hydroquinone under alkaline conditions reduced tetrazolium salts to formazan, which exhibited absorbance maxima at 440 nm. A linear relationship between the absorbance and viable cell density was obtained in the range of 1.0 x 10(5)-2.0 x 10(7) cells/ml for a sample solution. During the cultivation of yeast cells, the absorbance showed almost an anti-parallel change with that of glucose in yeast growth and fermentation, suggesting that the absorbance change reflected the vitality of yeast cells.