Activity and mechanisms of action of selected biocidal agents on Gram-positive and -negative bacteria

AIMS: This study investigates the antimicrobial activity and mode of action of two natural products, eugenol and thymol, a commonly utilized biostatic agent, triclocarban (TCC), and two surfactants, didecyldimethylammonium chloride (DDDMAC) and C10-C16 alkyldimethyl amine N-oxides (ADMAO). METHODS AND RESULTS: Methods used included: determination of minimum inhibitory concentrations (MICs), lethal effect studies with suspension tests and the investigation of sub-MIC concentrations on growth of E. coli, Staph. aureus and Ps. aeruginosa using a Bioscreen microbiological analyser. Leakage of intracellular constituents and the effects of potentiating agents were also investigated. Only DDDMAC was bactericidal against all of the organisms tested. Eugenol, thymol and ADMAO showed bacteriostatic and bactericidal activity, but not against Ps. aeruginosa. TCC was only bacteristatic against Staph. aureus, but like the other agents, it did affect the growth of the other organisms in the Bioscreen experiments. All of the antimicrobial agents tested were potentiated by the permeabilizers to some extent and leakage of potassium was seen with all of the agents except TCC. CONCLUSIONS: DDDMAC was bactericidal against all organisms tested and all compounds had some bacteriostatic action. Low level static effects on bacterial growth were seen with sub-MIC concentrations. Membrane damage may account for at least part of the mode of action of thymol, eugenol, DDDMAC and ADMAO. SIGNIFICANCE AND IMPACT OF THE STUDY: The ingredients evaluated demonstrated a range of bactericidal and bacteriostatic properties against the Gram-negative and -positive organisms evaluated and the membrane (leakage of intracellular components) was implicated in the mode of action for most (except TCC). Sub-MIC levels of all ingredients did induce subtle effects on the organisms which impacted bacterial growth, even for those which had no true inhibitory effects.     

A universal protocol for photochemical covalent immobilization of intact carbohydrates for the preparation of carbohydrate microarrays

A universal photochemical method has been established for the immobilization of intact carbohydrates and their analogues, and for the fabrication of carbohydrate microarrays. The method features the use of perfluorophenyl azide (PFPA)-modified substrates and the photochemical reaction of surface azido groups with printed carbohydrates. Various aldoses, ketoses, nonreducing sugars such as alditols, and their derivatives can be directly arrayed on the PFPA-modified chips. The lectin-recognition ability of arrayed mannose, glucose, and their oligo- and polysaccharides were confirmed using surface plasmon resonance imaging and laser-induced fluorescence imaging.     

Chemotaxis of cholera vibrios: a study method and the relation to different substances

The chemotaxis of V. cholerae in response to 56 different substances (amino acids, carbohydrates, salts, etc.) has been studied by the methods of visual observation and quantitative determination. Attractants, neutral substances and one repellent have been revealed. Adler's method (1973) has been modified with regard to the requirements for the working procedures in handling the causative agents of highly dangerous infections.     

Antibacterial Effects of Glycyrrhetinic Acid and Its Derivatives on Staphylococcus aureus

Staphylococcus aureus is a major pathogen in humans and causes serious problems due to antibiotic resistance. We investigated the antimicrobial effect of glycyrrhetinic acid (GRA) and its derivatives against 50 clinical S. aureus strains, including 18 methicillin-resistant strains. The minimum inhibitory concentrations (MICs) of GRA, dipotassium glycyrrhizate, disodium succinoyl glycyrrhetinate (GR-SU), stearyl glycyrrhetinate and glycyrrhetinyl stearate were evaluated against various S. aureus strains. Additionally, we investigated the bactericidal effects of GRA and GR-SU against two specific S. aureus strains. DNA microarray analysis was also performed to clarify the mechanism underlying the antibacterial activity of GR-SU. We detected the antimicrobial activities of five agents against S. aureus strains. GRA and GR-SU showed strong antibacterial activities compared to the other three agents tested. At a higher concentration (above 2x MIC), GRA and GR-SU showed bactericidal activity, whereas at a concentration of 1x MIC, they showed a bacteriostatic effect. Additionally, GRA and GR-SU exhibited a synergistic effect with gentamicin. The expression of a large number of genes (including transporters) and metabolic factors (carbohydrates and amino acids) was altered by the addition of GR-SU, suggesting that the inhibition of these metabolic processes may influence the degree of the requirement for carbohydrates or amino acids. In fact, the requirement for carbohydrates or amino acids was increased in the presence of either GRA or GR-SU. GRA and GR-SU exhibited strong antibacterial activity against several S. aureus strains, including MRSA. This activity may be partly due to the inhibition of several pathways involved in carbohydrate and amino acid metabolism.     

中药鹿藿的抑菌实验研究

研究发现中药鹿藿在体外可瞬间抑制人类精子的运动。为了研究中药鹿藿的抑菌作用,本实验对几种女性生殖道常见致病菌和条件致病菌进行了体外抑菌实验,发现鹿藿对金黄色葡萄球菌、淋病奈瑟氏菌和大肠杆菌有抑制作用,并可抑制常见耐药菌株的生长。金黄色葡萄球菌的MIC折合生药为31.25mg/mL,大肠杆菌的MIC250mg/mL,淋球菌的MIC为31.25mg/mL。结果表明中药鹿藿在能有效抑制精子运动的浓度可以抑制某些男、女性生殖道常见细菌的生长。     

Ion chromatography of azide in pharmaceutical protein samples with high chloride concentration using suppressed conductivity detection

Methods based on reversed-phase liquid chromatography with UV detection of 4-nitrobenzoyl- or 3,5-dinitrobenzoyl azide derivatives lack in accuracy and stability of derivatives to be applied for azide determination in pharmaceutical protein samples with high sodium chloride concentrations. This paper describes a sensitive and selective ion chromatographic method, with simple sample preparation and suppressed conductivity detection, developed for trace determination of azide in protein samples containing sodium chloride in concentrations as high as 11.6 g L(-1). Anion exchange stationary phase with quaternary alkyl amine functional groups and gradient elution with sodium hydroxide enabled good resolution of anions with similar retention times: azide, bromide and nitrate, as well as chloride whose retention time was shorter than azide's. Anions with high affinity to stationary phase (phosphate and citrate) were also eluted within acceptable analysis time of 32 min. The stability of sample solutions and the method selectivity, accuracy, precision and sensitivity satisfied the validation criteria of international organizations competent for pharmaceutical industry. The detection and quantitation limit ranges of sodium azide in protein samples were 0.007-0.02 mg L(-1) and 0.02-0.06 mg L(-1), respectively. Both limits increased with the concentration of sodium chloride.     

Quenching of carbohydrate reactions by azide

Sodium azide, at concentrations that prevent development of microorganisms, interferes with the anthrone and phenol sulfuric acid reactions for carbohydrates, and with the glucose oxidase reaction. It does not affect copper reduction and the ortho-toluidine reaction.     

Exploitation of β-glycosyl azides for the preparation of α-glycosynthases

An interesting approach for the chemo-enzymatic synthesis of carbohydrates is the use of glycosynthases, a class of mutant glycosidases derived from β-glycoside hydrolases obtained by replacement of the active site nucleophile with a non-nucleophilic residue. However, the scarcity of α-glycosynthases has so far hampered access to the synthesis of a large class of oligosaccharides of biotechnological interest. We review here a new glycosynthetic methodology for the production of two retaining α-fucosynthases and an α-galactosynthase exploiting β-glycosyl azide derivatives. The general applicability of this approach, which opens new perspectives in the use of azide derivatives for the production of novel α-glycosynthases, is discussed.     

A Method for the Determination of Antimicrobial Properties of Treated Fabrics

A method has been presented for evaluating the effectiveness of fabrics treated with antimicrobial agents to resist growth of contaminating organisms. The method consists of direct inoculation of the fabric with suitable test organisms and the subsequent enumeration of growing colonies which develop when the fabric is implanted in a nutrient medium.The method is sensitive, versatile, easy to perform, highly reproducible, and realistically evaluates the practical effectiveness of residual antimicrobial agents on fabrics.Data are given which demonstrate the sensitivity of the method to small differences in antimicrobial concentrations. The versatility of the method is shown by the variety of antimicrobial agents and organisms which have been utilized. Both bacteriostatic and bactericidal activity can be evaluated.     

Application of improved iodine-azide procedure for the detection of thiouracils in blood serum and urine with planar chromatography

The application of iodine-azide reaction for the determination of thiouracils in thin-layer chromatography and high-performance thin-layer chromatography is described. The developed plates were sprayed with a freshly prepared mixture of sodium azide, adjusted to a proper pH, and starch solution, and exposed to iodine vapour for 5 s. The detection limits were established at pmol level. The factors depending on the detection limits were described. A comparison of iodine-azide tests reaction with other procedures is presented. The developed method was applied to detection of thiouracils in blood serum and urine. The possibility of detection of a thiouracils mixture was demonstrated.     

Fluorometric determination of sialic acids using malononitrile in weakly alkaline media and its application to postcolumn labeling in high-performance liquid chromatography

A sensitive method for determination of sialic acids by monitoring the fluorescence produced with malononitrile in borate buffer has been established. Measurement of the fluorescence intensity of the reaction mixture at 430 nm with irradiation at 360 nm allowed determination of 3-60 nmol of sialic acids with high reproducibility. A few amino sugars and deoxy sugars, as well as catecholamines reacted with this reagent; however other carbohydrates, amino acids, amines, aldehydes, and carboxylic acids including alpha-keto acids, etc., showed little reactivity. This method was successfully applied to postcolumn fluorescence labeling of sialic acids in high-performance liquid chromatography.     

Identification and characterization of some bacterial membrane sulfhydryl groups which are targets of bacteriostatic and antibiotic action

Covalent modification of sulfhydryl groups which become sensitive toward sulfhydryl agents during germination of Bacillus cereus spores exerts a profound bacteriostatic effect, resulting in outgrowth inhibition. The modified spore components are membrane species of 13,000, 28,000, and 29,000 daltons. Detergent disruption of the membrane inactivated the sulfhydryl groups. A highly sigmoid inhibition curve (n = 11.8) with diamide suggested the participation of closely neighboring sulfhydryl groups. Substate and substrate analogs of the lactose and dicarboxylic acid permeases protected the sulfhydryl groups against modification. Nisin, a 34-residue peptide antibiotic, inhibited spore outgrowth and sulfhydryl modification at a concentration of about 0.1 microM. Since these sulfhydryl groups have been implicated as involved with the bacteriostatic action of nitrite, substances directed toward them may be a useful new class of bacteriostatic agents and antibiotics.     

Accumulation of Golgi-processed secretory proteins in an organelle of high density upon reduction of ATP concentration in rat hepatocytes.

We have previously shown that when rat hepatocytes are incubated with 4 mM azide, which reduces the intracellular ATP concentration to about 30% of its normal level, secretory proteins are reversibly arrested within the cell. Analysis of haptoglobin after 150 min of azide incubation shows that its carbohydrates have been processed by Golgi enzymes (Persson, R., Ahlstr?m, E., and Fries, E. (1988) J. Cell Biol. 107, 2503-2510). Here, we have further characterized the site of arrest. Subcellular fractionation by density gradient centrifugation showed that albumin and haptoglobin fractionated like a marker for the endoplasmic reticulum. Localization of albumin by immunoelectron microscopy showed, however, that it occurred in flattened cisternae and that the endoplasmic reticulum was devoid of the protein. A possible explanation for these results is that the azide treatment blocks transport through the Golgi complex, leading to an accumulation of secretory proteins in a pre- or early Golgi compartment of high density. This compartment could contain sufficient amounts of Golgi enzymes to carry out the observed carbohydrate processing upon prolonged incubation or possibly acquire them as an effect of the azide treatment.     

Percolation of starch and soluble carbohydrates from plant tissue for quantitative determination with anthrone.

The technique of percolation was applied as an improvement over the usual technique of maceration for extraction of carbohydrates from small amounts of plant material. Soluble carbohydrates were extracted by percolation with 80% ethanol and starch was extracted by percolation with 35% perchloric acid. The course of percolation was studied and the technique was demonstrated to give reproducible results. The anthrone method was applied for starch determination. The influence by cellulose on the determination of starch, as well as, the influence of perchloric acid on the anthrone reaction was investigated. An analytical procedure based on the obtained results is given.     

Evaluation of antimicrobial interactions between chlorhexidine, quaternary ammonium compounds, preservatives and excipients.

The antimicrobial interactions of 49 combinations of chlorhexidine, quaternary ammonium compounds, preservatives and excipients were evaluated by the method of Berenbaum and the checkerboard titration method, with Staphylococcus aureus CIP 53154 and Escherichia coli CIP 54127 as test strains. MIC determinations were carried out as a preliminary step, and relative growth intensity was used to describe the bacteriostatic activity of surface-active agents (Amonyl 380 BA, Amonyl 671 SB). In the study of combinations, results were interpreted with Fractional Inhibitory Concentration indexes and represented by isobolograms. A fair correlation was shown between the method of Berenbaum and the checkerboard titration method. Combinations between chlorhexidine, cetrimonium bromide and benzalkonium chloride were synergistic or additive; combinations of antiseptics and preservatives were generally not antagonistic. The methods were also well adapted to the study of interactions involving surface-active agents, a critical problem in the formulation of topical antimicrobial agents.     

Evaluation of antimicrobial interactions between chlorhexidine, quaternary ammonium compounds, preservatives and excipients

The antimicrobial interactions of 49 combinations of chlorhexidine, quaternary ammonium compounds, preservatives and excipients were evaluated by the method of Berenbaum and the checkerboard titration method, with Staphylococcus aureus CIP 53154 and Escherichia coli CIP 54127 as test strains. MIC determinations were carried out as a preliminary step, and relative growth intensity was used to describe the bacteriostatic activity of surface-active agents (Amonyl 380 BA®, Amonyl 671 SB®). In the study of combinations, results were interpreted with Fractional Inhibitory Concentration indexes and represented by isobolograms. A fair correlation was shown between the method of Berenbaum and the checkerboard titration method. Combinations between chlorhexidine, cetrimonium bromide and benzalkonium chloride were synergistic or additive; combinations of antiseptics and preservatives were generally not antagonistic. The methods were also well adapted to the study of interactions involving surface-active agents, a critical problem in the formulation of topical antimicrobial agents.     

A spectrophotometric method for simultaneous determination of protein-bound hexoses and fucose with a mixture of L-cysteine and phenol

A very suitable spectrophotometric method for simultaneous determination of protein-bound hexoses and fucose is presented. A mixture of L-cysteine and phenol in sulfuric acid was used as reagent, whereas absorption measurements were carried out at two wavelengths, namely at 398 nm for fucose and 490 nm for hexoses determination. Optimum conditions for the application of the method were established, special attention being paid to the possible interference of fucose determination with that of hexoses, and vice versa. The proposed method was applied to the determination of hexoses and fucose in serum glycoproteins and seromucoids. The method was found to be very practical enabling a simultaneous determination of both kinds of carbohydrates; moreover, it was proved to be more sensitive and specific in comparison with methods commonly used for individual determination of fucose with L-cysteine and hexoses with phenol.     

Hydrogen Peroxide Formation by Cells Treated with a Tumor Promoter

To determine whether oxidants capable of DNA modification are produced by cells treated with tumor promoters, we adapted a fluorometric method to our needs. HeLa cells were preincubated with 2‘,7‘-dichlorofluorescin diacetate (DCFdAc), treated with various agents, sonicated. centrifuged and fluorescence of the oxidized product (DCF) was determined in supernatants. When cells were exposed to H₂O₂ in the presence of azide (catalase inhibitor) or o-phenanthroline (a lipophilic Fe chelator), an increase in fluorescence was observed. These results show that some Fe ions were interacting with the H₂O₂ which entered the cells, thus decreasing its levels available for oxidation of the substrate and potentially increasing formation of OH, known DNA-damaging species. Glutathione (GSH). which is present in cells in substantial amounts, was found to reduce DCF whereas azide counteracted GSH-mediated reduction.

Treatment of HeLa cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of DCFdAc and azide resulted in dose-and time-dependent formation of DCF. Even when cells were sonicated prior to incubation with TPA, DCF was formed at levels proportional to the number of cells as well as dose of TPA. Flow cytometry of TPA-treated cells confirmed these findings.

These results demonstrate that tumor promoters can cause oxidative activation of HeLa cells, which produce active oxygen species, most likely H₂O₂, that ultimately contribute to the formation of oxidized bases such as 5-hydroxymethyl uracil in cellular DNA. They also show that this fluorometric method can be utilized for determination of cellular H₂O₂ formation at nM concentrations.     

Biosensing based on surface plasmon resonance and living cells

We propose the combination of surface plasmon resonance (SPR) with living cells as a biosensing method. Our detection scheme is based on the premise that cellular activity induced by external agents is often associated with changes in cellular morphology, which in turn should lead to a variation of the effective refractive index at the interface between the cell membrane and the metal layer. We monitored surface plasmon resonance signals originating from a gold surface coated with cells on a custom apparatus after injection of various agents known to influence cellular activity and morphology. Specifically, we evaluated three types of stimulation: response to an endotoxin (lipopolysaccharides), a chemical toxin (sodium azide) and a physiological agonist (thrombin). A comparison with phase contrast microscopy reveals that SPR signal variations are associated with the induction of cell death for lipopolysaccharides treatment and a contraction of the cell body for sodium azide. Thrombin-induced cellular response shows a rapid decrease of the measured laser reflectance over 5min followed by a return to the original value. For this treatment, phase contrast micrographs relate the first phase of the SPR variation to cell contraction and increase of the intercellular gaps, whereas the recovery phase can be associated with a spreading of the cell on the sensing surface. Hence, the SPR signal is very consistent with the cellular response normally observed for these treatments. This confirms the validity of the biosensing method, which could be applied to a large variety of cellular responses involving shape remodeling induced by external agents.     

Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine.

Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity.