Inhibitory effect of biocides on the viable masses and matrices of Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

Bacteria and matrix are essential for the development of biofilms, and assays should therefore target both components. The current European guidelines for biocidal efficacy testing are not adequate for sessile microorganisms; hence, alternative discriminatory test protocols should be used. The activities of a broad range of biocides on Staphylococcus aureus and Pseudomonas aeruginosa biofilms were evaluated using such in vitro assays. Nearly all selected biocides showed a significant decrease in S. aureus biofilm viability, with sodium hypochlorite and peracetic acid as the most active biocides. Only hydrogen peroxide and sodium hypochlorite showed some inhibitory effect on the matrix. Treatment of P. aeruginosa biofilms was roughly comparable to that of S. aureus biofilms. Peracetic acid was the most active on viable mass within 1 min of contact. Isopropanol ensured a greater than 99.999% reduction of P. aeruginosa viability after at least 30 min of contact. Comparable to results with S. aureus, sodium hypochlorite and hydrogen peroxide markedly reduced the P. aeruginosa matrix. This study clearly demonstrated that despite their aspecific mechanisms of action, most biocides were active only against biofilm bacteria, leaving the matrix undisturbed. Only hydrogen peroxide and sodium hypochlorite were active on both the biofilm matrix and the viable mass, making them the better antibiofilm agents. In addition, this study emphasizes the need for updated and standardized guidelines for biofilm susceptibility testing of biocides.

Evaluation of hydrogen peroxide-based disinfectants in a new resazurin microplate method for rapid efficacy testing of biocides.

AIMS: Development of the resazurin microplate method (RMM) as a novel test system for the evaluation of the antimicrobial activity of antiseptics and disinfectants. The validated RMM was subsequently applied for the evaluation of hydrogen peroxide (H(2)O(2)) and stabilized H(2)O(2) combination products. METHODS AND RESULTS: The European Committee for Standardization prescribes the plate count challenge test (PCCT) for antiseptic and disinfectant efficacy testing. This protocol was adapted to a microplate-based assay, using resazurin as viability indicator. The RMM was as accurate as the PCCT, had an identical detection limit and showed high intermediate precision. Using the validated RMM, it was shown that H(2)O(2) combined with silver possessed a higher bactericidal and fungicidal activity compared to native H(2)O(2) with and without glycerol. CONCLUSIONS: Validation showed that the RMM may replace the PCCT. When applying the RMM, H(2)O(2) combined with silver was clearly a more potent disinfectant compared to H(2)O(2) in killing bacteria and fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: The RMM is easier to use for antimicrobial efficacy testing of antiseptics and disinfectants. As the RMM is in accordance with the norms of the European Committee for Standardization, it may become a more cost-effective alternative to the more laborious PCCT reference method. H(2)O(2) with silver may replace native H(2)O(2) to increase overall disinfection efficiency.

A new colorimetric microtitre model for the detection of Staphylococcus aureus biofilms.

AIMS: Research on biofilms requires validated quantitative models that focus both on matrix and viable bacterial mass. In this study, a new microplate model for the detection of Staphylococcus aureus biofilms was developed. METHODS AND RESULTS: Dimethyl methylene blue (DMMB) dye was used to quantify biofilm matrix colorimetrically. Initially developed for the detection of glycosaminoglycans, the DMMB protocol was optimized for S. aureus biofilm research. In addition, the redox indicator resazurin was used to determine the viable bacterial biofilm burden. CONCLUSION: A new, simple and reproducible microplate test system based on DMMB and resazurin, offering a reliable differentiation between biofilm matrix and cellular activity, was developed and validated for the detection of S. aureus biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: The DMMB-resazurin microtitre plate model is a valuable tool for high capacity screening of biocides and for the development of synergistic mixtures of biocides, destroying both biofilm matrix and bacteria.

Challenges and pitfalls in antioxidant research.

Over the last decade, much research has focused on the potential health benefits of antioxidants and indeed many synthetic and natural compounds have been evaluated for their antioxidant profile. However, in several studies only a limited number of assays, often poorly validated, are used and the techniques available frequently lack specificity. These limitations may incorrectly influence the results. This review will therefore focus on several pitfalls that may emerge in vitro and in vivo antioxidant research. First, different in vitro techniques to determine antioxidant potential are discussed, including radical scavenging assays and fingerprinting methods. As a rule, a panel of different assays is indispensable to characterize and establish in vitro antioxidant activity. Furthermore, as problems of absorption, distribution, metabolism and excretion are only accounted for by in vivo studies, the need for in vivo antioxidant research is pointed out. Several methods to characterize the in vivo activity of antioxidants, including major drawbacks and pitfalls of some assays, have been discussed. The availability of both a representative "oxidative stress" animal model and a battery of well-validated assays to assess the broad diversity of oxidative damage and antioxidative defence parameters, are crucial for antioxidant research in vivo.

Effect of oral intake of choline-stabilized orthosilicic acid on hair tensile strength and morphology in women with fine hair.

The appearance of hair plays an important role in people's overall physical appearance and self-perception. Silicon (Si) has been suggested to have a role in the formation of connective tissue and is present at 1-10 ppm in hair. Choline-stabilized orthosilicic acid ("ch-OSA") is a bioavailable form of silicon which was found to improve skin microrelief and skin mechanical properties in women with photoaged skin. The effect of ch-OSA on hair was investigated in a randomized, double blind, placebo-controlled study. Forty-eight women with fine hair were given 10 mg Si/day in the form of ch-OSA beadlets (n = 24) or a placebo (n = 24), orally for 9 months. Hair morphology and tensile properties were evaluated before and after treatment. Urinary silicon concentration increased significantly in the ch-OSA supplemented group but not in the placebo group. The elastic gradient decreased in both groups but the change was significantly smaller in the ch-OSA group (-4.52%) compared to placebo group (-11.9%). Break load changed significantly in the placebo group (-10.8%) but not in the ch-OSA supplemented group (-2.20%). Break stress and elastic modulus decreased in both groups but the change was smaller in the ch-OSA group. The cross sectional area increased significantly after 9 months compared to baseline in ch-OSA supplemented subjects but not in the placebo group. The change in urinary silicon excretion was significantly correlated with the change in cross sectional area. Oral intake of ch-OSA had a positive effect on tensile strength including elasticity and break load and resulted in thicker hair.

Effect of oral intake of choline-stabilized orthosilicic acid on skin, nails and hair in women with photodamaged skin.

Chronic exposure of the skin to sunlight causes damage to the underlying connective tissue with a loss of elasticity and firmness. Silicon (Si) was suggested to have an important function in the formation and maintenance of connective tissue. Choline-stabilized orthosilicic acid ("ch-OSA") is a bioavailable form of silicon which was found to increase the hydroxyproline concentration in the dermis of animals. The effect of ch-OSA on skin, nails and hair was investigated in a randomized, double blind, placebo-controlled study. Fifty women with photodamaged facial skin were administered orally during 20 weeks, 10 mg Si/day in the form of ch-OSA pellets (n=25) or a placebo (n=25). Noninvasive methods were used to evaluate skin microrelief (forearm), hydration (forearm) and mechanical anisotropy (forehead). Volunteers evaluated on a virtual analog scale (VAS, "none=0, severe=3") brittleness of hair and nails. The serum Si concentration was significantly higher after a 20-week supplementation in subjects with ch-OSA compared to the placebo group. Skin roughness parameters increased in the placebo group (Rt:+8%; Rm: +11%; Rz: +6%) but decreased in the ch-OSA group (Rt: -16%; Rm: -19%; Rz: -8%). The change in roughness from baseline was significantly different between ch-OSA and placebo groups for Rt and Rm. The difference in longitudinal and lateral shear propagation time increased after 20 weeks in the placebo group but decreased in the ch-OSA group suggesting improvement in isotropy of the skin. VAS scores for nail and hair brittleness were significantly lower after 20 weeks in the ch-OSA group compared to baseline scores. Oral intake of ch-OSA during the 20 weeks results in a significant positive effect on skin surface and skin mechanical properties, and on brittleness of hair and nails.

4'-Hydroxy-3-methoxyflavones with potent antipicornavirus activity.

4'-Hydroxy-3-methoxyflavones are natural compounds with known antiviral activities against picornaviruses such as poliomyelitis and rhinoviruses. In order to establish a structure-activity relationship a series of analogues were synthesized, and their antiviral activities and cytotoxicities were compared with those of flavones from natural origin. The 4'-hydroxyl and 3-methoxyl groups, a substitution in the 5 position and a polysubstituted A ring appeared to be essential requirements for a high activity. The most interesting compound was 4',7-dihydroxy-3-methoxy-5,6-dimethylflavone possessing in vitro TI99 values of greater than 1000 and greater than 200 against poliovirus type 1 and rhinovirus type 15, respectively. This compound was also active against other rhinovirus serotypes (2, 9, 14, 29, 39, 41, 59, 63, 70, 85, and 89) tested, having MIC50 values ranging from 0.016 to 0.5 micrograms/mL. Finally in contrast to quercetin it showed to be not mutagenic in concentrations up to 2.5 mg in the Ames test.

Lack of evidence for strand-specific inhibition of poliovirus RNA synthesis by 3-methylquercetin.

Buffalo Green Monkey cells were infected with poliovirus-1 in the presence of 3-methylquercetin or guanidine, and the formation of positive- and negative-strand viral RNA was monitored using single-stranded RNA probes. Both 3-methylquercetin and guanidine prevented the formation of plus-strand as well as minus-strand viral RNA, although, due to the high multiplicity of infection used, a high number of genomic viral RNA was always present in the cells.

The poliovirus-induced shut-off of cellular protein synthesis persists in the presence of 3-methylquercetin, a flavonoid which blocks viral protein and RNA synthesis.

In poliovirus-infected cells, the viral protein and RNA synthesis were severely reduced, provided 3-methylquercetin was present between 1 and 2 h post-infection. Under these conditions, the virally induced host shut-off remained in effect. On the other hand, in uninfected HeLa cells, protein and RNA synthesis was inhibited only slightly by 3-methylquercetin. The inhibition of poliovirus cytopathogenicity in Vero cells by 3-methylquercetin exhibited a similar time dependence.

Selenium and the growth of Haemophilus ducreyi.

One of the growth media in current use for Haemophilus ducreyi comprises Mueller Hinton agar, chocolatised horse blood, serum and IsoVitalex (BBL). For a better understanding of growth factors, attempts were made to simplify this complex medium. The horse blood was replaced by haemin (200 micrograms/ml), the serum by albumin (0.2%), and IsoVitalex was substituted only by L-glutamine 0.01%. Most of the strains grew, but when selenium ions were added in a concentration of 3.25 x 10(-3) micrograms/ml, growth was stimulated and became more luxuriant than growth on conventional media.

Mutagenicity of pyrrolizidine alkaloids in the Salmonella typhimurium/mammalian microsome system.

The mutagenicity of a series of pyrrolizidine alkaloids, and of extracts from several Italian Senecio species containing pyrrolizidine alkaloids, including S. inaequidens, S. fuchsii and S. cacaliaster, were tested using the Salmonella typhimurium/mammalian microsome system. Retrorsine, senecivernine, seneciphylline and the Senecio extracts showed a weakly mutagenic activity.

Inhibition of the multiplication of enveloped and non-enveloped viruses by glucosamine.

Glucosamine can inhibit the development of viral cytopathogenic effect and the production of infective viral particles of both enveloped and non-enveloped viruses. The extent of antiviral activity is dependent on drug concentration, composition of the culture medium and type of cell host.

Comparative study of eight well-known polyphenolic antioxidants.

Eight antioxidants from five different polyphenolic classes (cinnamic acids, benzoic acids, flavonoids, proanthocyanidins and stilbenes), and the water-soluble vitamin E derivative trolox were examined for their antioxidant activity in-vitro. In addition, the compounds were tested for their cytotoxicity on growing fibroblasts and their inhibition of the classical pathway of the complement system. Procyanidin C1 was shown to be a good scavenger of both DPPH(*) and HO(*), and a strong inhibitor of lipid peroxidation and the classical pathway of the complement system. Consequently, procyanidin C1 was classified as the most promising antioxidant in-vitro of all compounds tested. In contrast, genistein exhibited a very low antioxidant activity in both the lipid peroxidation and the DPPH(*) scavenging assay, a high cytotoxicity and a low complement-inhibiting activity.

Radical scavenging and xanthine oxidase inhibitory activity of phenolic compounds from Bridelia ferruginea stem bark.

Bridelia ferruginea Benth. (Euphorbiaceae) is a subtropical medicinal plant widely used in traditional African medicine against various diseases, including rheumatic pains. Seven of its constituents (3-O-methylquercetin (1), 3,7,3',4'-tetra-O-methylquercetin (rutisin, 2), myricetin (3), 3',4',5'-tri-O-methylmyricetin (ferrugin, 4), 3,3',4',5'-tetra-O-methylmyricetin (5), quercetin 3-O-glucoside (6), and a biflavanol gallocatechin-[4'-O-7]-epigallocatechin (7)) have been evaluated in-vitro in the xanthine-xanthine oxidase enzymatic system for inhibition of xanthine oxidase and radical scavenging activity. Results indicated that compounds 1, 3, 4 and 6 exhibited, at different levels, xanthine oxidase inhibiting and superoxide scavenging activity at micromolar concentrations, whereas compound 7 showed scavenging activity only. Compounds 2 and 5 were inactive in both cases. Study of the structure-activity relationship demonstrated that for flavonoids the xanthine oxidase inhibitory activity was reduced by methylation of the hydroxyl functionality at C-3 and in rings A and B. These results may partly explain and support the use of B. ferruginea stem bark for the treatment of rheumatic pains in traditional medicine.

Selenium in environmental and drinking waters of Belgium.

The tetravalent and hexavalent selenium content of water samples can be determined by conventional energy-dispersive X-ray fluorescence after different preconcentration steps. Selenium values of nearly three-hundred different environmental and drinking water samples in Belgium are reported. The results are quite low, ranging from the detection limits up to 1 microgram 1-1. The concentration levels are compared to literature data and the speciation is discussed. The contribution of drinking water to the daily intake of selenium in Belgium ranges from less than 0.2 to 5%.

Can ethnopharmacology contribute to the development of antiviral drugs?

In recent years, many compounds having potent antiviral activity in cell cultures and in experimental animals have been detected, but only a few have been approved by Western health authorities for clinical use. Nevertheless, some of these compounds are currently undergoing either preclinical or clinical evaluation, and perspectives for finding new interesting antiviral drugs are promising. Among these antiviral substances are several natural compounds isolated from plants used in traditional medicine including polysaccharides, flavonoids, terpenes, alkaloids, phenolics and amino acids. Some of these plant compounds exhibit a unique antiviral mechanism of action and are good candidates for further clinical research. What follows is a brief summary of the selection methods of plants for antiviral screening and in vitro and in vivo assays, which are currently used for detecting this activity in plant extracts. The importance of the plant kingdom as a source of new antiviral substances will be illustrated by presenting a survey on plant-derived antirhinovirus and anti-HIV agents.

Screening of seven selected Rwandan medicinal plants for antimicrobial and antiviral activities.

Aqueous EtOH (80%) extracts of seven plants used by Rwandan traditional healers to treat infections, were screened for antibacterial, antifungal, and antiviral activities. Only two of the selected plants showed a true antiviral activity against herpes simplex virus type 1, while all of them exhibited virucidal properties against several enveloped viruses including herpes simplex, measles, Semliki forest, and vesicular stomatitis viruses. Four plants were diversely active against gram-positive bacteria, two of these showing bactericidal effect against the acid-fast Mycobacterium fortuitum. None of the selected plants was active against gram-negative bacteria or the yeast Candida albicans. From a bioassay-guided fractionation procedure using herpes simplex virus type I as the target model, a virucidal mixture, the maesasaponin mixture A, was isolated from the MeOH extract of Maesa lanceolata. The maesasaponin mixture A exhibited a virucidal activity against herpes simplex types 1 and 2, and vesicular stomatitis viruses.

Further evaluation of Rwandan medicinal plant extracts for their antimicrobial and antiviral activities.

A total of 45 Rwandan plant extracts, belonging to 37 different plant species out of 21 families, were investigated for their antibacterial, antifungal, and antiviral properties. The plants were selected on the base of their ethnomedicinal use against infections and autoimmune diseases. From all the plant extracts tested, only Clematis hirsuta (leaves) showed a pronounced antifungal activity against Candida albicans and the dermatophytes Trichophyton rubrum, Epidermophyton floccosum, and Microsporum canis. Seven plant extracts showed a high antiviral activity against the DNA-virus Herpes simplex type 1, while five and three plant extracts were highly active against the RNA-viruses Coxsackie and Polio, respectively. Only Macaranga kilimandscharica (leaves) showed an interesting anti-measles activity, whereas Eriosema montanum (leaves) and Entada abyssinica (leaves) were highly active against Semliki forest virus. Some plant extracts showed an antibacterial activity against Gram-positive bacteria and Mycobacterium fortuitum, but none of them were active against the Gram-negative bacteria tested.

Screening of hundred Rwandese medicinal plants for antimicrobial and antiviral properties.

A series of 100 Rwandese medicinal plants (267 plant extracts), used by traditional healers to treat infections, were screened for antibacterial, antifungal and antiviral properties. The results of the testing showed that 45% were active against Staphylococcus aureus, 2% against Escherichia coli, 16% against Pseudomonas aeruginosa, 7% against Candida albicans, 80% against Microsporum canis and 60% against Trichophyton mentagrophytes. Not less than 27% of the plant species exhibited prominent antiviral properties against one or more test viruses, more specifically 12% against poliomyelitis, 16% against coxsackie, 3% against Semliki forest, 2% against measles and 8% against herpes simplex virus.

Anti-microbial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants.

Selected plants having a history of use in Polynesian traditional medicine for the treatment of infectious disease were investigated for anti-viral, anti-fungal and anti-bacterial activity in vitro. Extracts from Scaevola sericea, Psychotria hawaiiensis, Pipturus albidus and Eugenia malaccensis showed selective anti-viral activity against Herpes Simplex Virus-1 and 2 and Vesicular Stomatitis Virus. Aleurites moluccana extracts showed anti-bacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, while Pipturus albidus and Eugenia malaccensis extracts showed growth inhibition of Staphylococcus aureus and Streptococcus pyogenes. Psychotria hawaiiensis and Solanum niger inhibited growth of the fungi Microsporum canis, Trichophyton rubrum and Epidermophyton floccosum, while Ipomoea sp., Pipturus albidus, Scaevola sericea, Eugenia malaccensis, Piper methysticum, Barringtonia asiatica and Adansonia digitata extracts showed anti-fungal activity to a lesser extent. Eugenia malaccensis was also found to inhibit the classical pathway of complement suggesting that an immunological basis for its in vivo activity was identified. This study has confirmed some of the ethnobotanical reports of Hawaiian medicinal plants having curative properties against infections using biological assays in vitro.