Visual analysis of vaginal cell binding and retention of a gynecological douche.

AIM: Gynecological douches may contain various molecules that need to cover and be retained by cutaneous and mucosal cells if they are to act efficaciously in treating local conditions. The aim of this study was to investigate the possibility of directly visualising the ability of a commercial medical gynecological douche to bind to, and be retained by human vaginal cells. METHODS: The commercial gynecological douche under study was "Saugella Attiva douche", bought at local chemist. The vaginal epithelial cells were obtained from healthy, non-pregnant, regularly menstruating women aged 24-52 years. The cells were obtained from the mucosal surface of the mid-vaginal wall by means of gentle scraping with a sterile spatula. Ferric oxide particles and Escherichia coli were used as inorganic and organic markers in order to visualize the adherence of the transparent thin film of a gynecological douche to human vaginal cells by means of Nomarski interference contrast microscopy and scanning electron microscopy. RESULTS: Both markers made it possible to clearly visualize the binding and retention of the transparent thin layer of the douche also at the dilution 1:2 and 1:4. CONCLUSIONS: The fact that the douche can be locally retained is useful because its formulation contains thymol and eugenol, which are known to have antibacterial, antifungal and antioxidant effects but need a period of contact before they act fully.

Eugenol and thymol, alone or in combination, induce morphological alterations in the envelope of Candida albicans.

The envelope of Candida albicans, with its outermost array of macromolecules protruding towards the environment, is pivotal to the expression of major virulence factors such as adhesiveness, and the morphological transition to hyphal form. We tested the anticandidal activity of eugenol, main component of clove oil, and thymol, main component of thyme oil, alone or in combination, by investigating their ability to interfere with the architecture of the envelope of C. albicans. Both molecules alterated the morphogenesis of the envelope, but the effects of thymol were more pronounced than those of eugenol. Certain combinations of the two molecules led to a synergistic effect, which is interesting in the view of potentiating their inhibition of C. albicans colonisation and infectiousness.

The post-antibiotic effects of rokitamycin (a 16-membered ring macrolide) on susceptible and erythromycin-resistant strains of Streptococcus pyogenes.

The post-antibiotic effects (PAEs) on susceptible and erythromycin-resistant strains of Streptococcus pyogenes (M phenotype and inducibly resistant) of rokitamycin and erythromycin were investigated in vitro using microbiological impedance measurement. Exposure of susceptible S. pyogenes strains to 1/4, 1/2, 1 and 2 MIC erythromycin and rokitamycin resulted in PAEs of rokitamycin in the same order of magnitude as those of erythromycin and that were dose dependent. The duration of rokitamycin PAEs in erythromycin-resistant S. pyogenes strains (M phenotype and those with inducible resistance) were comparable with those observed in susceptible strains. This was not the case for erythromycin. The investigation showed that a 16-membered ring macrolide such as rokitamycin has different PAEs from those of a 14-membered ring macrolide such as erythromycin. They also indicated that, as the PAEs of rokitamycin on the M phenotype and inducible resistant strains were comparable with those on susceptible strains, no re-evaluation of therapeutic dosing regimens was required.

Gemifloxacin: effects of sub-inhibitory concentrations on various factors affecting bacterial virulence.

This study investigated the ability of sub-MICs of gemifloxacin to interfere with the bacterial virulence parameters of adhesiveness, haemagglutination, hydrophobicity and motility, as well as their interactions with host neutrophilic defences such as phagocytosis, killing and respiratory bursts. The adhesiveness of both Escherichia coli and Staphylococcus aureus was significantly reduced to a subinhibitory concentration of 1/32 MIC. Indirect fimbriation parameters, such as hydrophobicity and haemagglutination were significantly reduced at a concentration of 1/8 MIC, as was migration (swarming). Phagocytosis and the respiratory burst measured by means of chemiluminescence were not affected, but killing was significantly increased from 1/2 to 1/8 MIC. The interpolation of these pharmacodynamic findings with pharmacokinetic curves indicates that sub-MIC concentrations of gemifloxacin can prolong antimicrobial effects on virulence determinants up to 27 h after the antimicrobial concentration has fallen below the MIC value.

Morphostructural damage and the inhibition of bacterial adhesiveness of Staphylococcus aureus and Moraxella catarrhalis induced by moxifloxacin.

The aim of this study was to investigate the ability of moxifloxacin to interfere with the mechanism of bacterial adhesion and disrupt the morphological and structural integrity of bacteria. Three Staphylococcus aureus and three Moraxella catarrhalis strains were grown in the presence of 1/2-1/128 minimum inhibitory concentration (MIC) serial dilutions and incubated with human epithelial cells. A significant decrease in adhesion was observed from 1/2 MIC to 1/64 MIC for S. aureus, and from 1/2 MIC to 1/16 MIC for M. catarrhalis. The use of atomic force microscopy, a new technique capable of revealing surface structures in three-dimensional detail and at very high resolution, showed the rapid onset and time course of the sequence of disruptive morphostructural events following the incubation of both S. aureus and M. catarrhalis with sub-MICs of moxifloxacin. Our findings suggest that less than conventional MIC moxifloxacin concentrations may be effective in reducing bacterial adhesiveness and structural integrity on which the maintenance of bacterial activity depends.

Interference of sub-inhibitory concentrations of gatifloxacin on various determinants of bacterial virulence.

The physico-chemical characteristics of the molecular array on the outermost surface of bacteria modulate various bacterial functions which, when expressed in the human environment, constitute important determinants of bacterial virulence. The present study investigated the ability of subinhibitory concentrations of gatifloxacin to interfere with various virulence determinants of Escherichia coli and with the adhesiveness of Staphylococcus aureus. The adhesiveness of S. aureus and E. coli to human epithelial cells was inhibited at gatifloxacin concentrations down to 1/32 and 1/64 the minimum inhibitory concentration (MIC). Sub-MICs of gatifloxacin down to 1/8-MIC significantly reduced hemagglutination and hydrophobicity, which are correlated with fimbriation and provide clues relating to the physico-chemical characteristics of the outer surface of bacteria. Swarming (motility) was reduced at concentrations down to 1/8 MIC. Phagocytosis was not affected but killing significantly increased from 1/8 to 1/2 MIC. The respiratory bursts of neutrophils investigated by a chemiluminescence procedure were not modified. The interpolation of these pharmacodynamic findings with pharmacokinetic curves indicates that the effect of sub-MIC concentrations of gatifloxacin can engender activity, prolonging antimicrobial effects on virulence determinants over 30 hours after the antimicrobial concentration has fallen below the MIC.

The combination of the SH metabolite of erdosteine (a mucoactive drug) and ciprofloxacin increases the inhibition of bacterial adhesiveness achieved by ciprofloxacin alone.

Exposure to ciprofloxacin and other fluoroquinolone antibiotics at less than minimum inhibitory concentrations (MICs) reduces the production of some of the factors that contribute to bacterial virulence, particularly bacterial adhesiveness. Once metabolized, erdosteine (a mucoactive drug) produces an active metabolite (Met I) with a reducing sulfhydryl group that is capable of opening the disulfide bonds present in tracheobronchial mucins and pilins, a protein of bacterial fimbriae (adhesins). This induces stereoconformational changes that interfere with the binding of bacterial adhesins to the receptors on mucosal cells. The combination of 5 and 10 micrograms/ml of Met I and 1/4, 1/8, 1/16 MICs of ciprofloxacin potentiated the inhibition of Staphylococcus aureus and Escherichia coli adhesiveness to human mucosal cells in comparison with ciprofloxacin alone. This finding opens up an interesting new possibility for interfering with bacterial adhesiveness and the resulting virulence by combining antibiotics with agents devoid of antibacterial activity.

The SH-metabolite I of erdosteine, a mucolytic drug, enhances the inhibitory effect of salbutamol on the respiratory burst of neutrophils.

Reactive oxygen species (ROS) are a common denominator of airway inflammation associated with chronic obstructive pulmonary disease (COPD) and asthma, as well as with less frequent lung diseases such as idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS) and cystic fibrosis (CF). The most frequently administered drugs used to treat these diseases are bronchodilators, antioxidant/antiphlogistic agents and mucoactive drugs. The metabolization of the mucoactive drug erdosteine produces an active metabolite (Met I) with a reducing SH group. In addition to its mucolytic action, Met I also has useful antioxidant activity. The various activities of beta 2-agonists include their ability to reduce the respiratory burst of neutrophils and the subsequent release of ROS. beta 2-Agonists and mucoactive drugs may be administered to the same patients during the treatment of lung diseases. The aim of this study was to investigate the ability of Met I to potentiate the activity of salbutamol in inhibiting the in vitro respiratory burst of neutrophils by means of chemiluminescence. The combination of Met I 5 and 10 micrograms/ml with salbutamol 10(-5), 10(-6) and 10(-7) M led to a significant reduction in respiratory bursts when the neutrophils were stimulated with the soluble stimulant N-formyl-methionylleucyl-phenylalanine (fMLP). The combinations of the two drugs that reduced the respiratory bursts when a particulate stimulus (Candida albicans) was used were those containing 10(-5) M of salbutamol. The reasons for this different behavior remain unclear and raise questions about the specific roles, sites and mechanisms of action of the different types of stimulation undergone by the respiratory airways.

Inhibitory activity of thymol against the formation and viability of Candida albicans hyphae.

As the capacity of Candida albicans to produce hyphae is considered an important virulence factor in the pathogenesis of candiasis, the aim of this study was to investigate whether thymol, the major component of thyme oil, can interfere with the filamentous forms of Candida albicans and their viability. The morphological transition from yeasts to filamentous forms was investigated by analysing the morphological index (MI), which classifies the differentiated forms and blastoconidia; viability was investigated by means of fluorescence microscopy using a new SYTO-9 and propidium iodide method previously used to stain only blastoconidia. Without thymol, there was an average of 94.00 +/- 3.06% hyphal forms. After 6 h of incubation with 1x MIC (125 microg ml(-1)), 1/2x MIC and 1/4x MIC of thymol, filamentation was, respectively, 14.33 +/- 8.25%, 28.33 +/- 7.17% and 45.67 +/- 8.09% in comparison with control (all statistically significant). In the absence of thymol, viable cells accounted for an average of 93.00 +/- 4.00% whereas, after 6 h of incubation with 1x MIC, 1/2x MIC and 1/4x MIC of thymol, the presence of 54.33 +/- 1.86%, 29.00 +/- 3.61% and 23.00 +/- 2.52% of yellow-orange coloured forms indicated damaged membranes and reduced viability. Our findings show that thymol interferes with the formation and viability of hyphae. This can be attributed to the characteristics of thymol disturbing Candida cell membranes and metabolism, probably by affecting fungal cell-wall synthesising enzymes.