Release of antiseptics from the aqueous compartments of a w/o/w multiple emulsion.

A w/o/w multiple emulsion drug carrier system has been developed for local vaginal therapy. To improve its efficacy and to extend the antimicrobial spectrum activity of benzalkonium chloride (CBZ), which is introduced in the external aqueous phase, chlorhexidine digluconate (CHD) was added to the internal aqueous phase of the multiple emulsions. The minimal bactericidal concentrations (MBC) for the association of CHD and CBZ in emulsion were determined towards Escherichia coli and Staphylococcus aureus. The main release mechanism considered for the CHD encapsulated in the inner phase was a swelling-breakdown phenomenon which followed dilution of the emulsion under hypo-osmotic conditions. In order to demonstrate this release, the bactericidal effect of multiple emulsions undiluted and diluted 1-5 and 1-10 in hypo-osmotic conditions at two CHD concentrations was evaluated. To validate and quantify this release, rheological and release kinetics studies were used. The bactericidal activity of combination CBZ-CHD in the emulsion was synergistic on the two bacterial strains and the release of encapsulated CHD in the internal phase was obtained following its dilution in hypo-osmotic conditions. Vaginal administration could be carried out following dilution at 1-5 in sterile water for multiple emulsions containing the lower concentration of CHD.

Synthesis and Crystal Structure of Diaqua(1,10-Phenanthroline-N,N')(Thiosulfato-O,S)Manganese(II). Biological Properties.

The synthesis of diaqua(1,10-phenanthroline-N,N')(thiosulfato-O,S)manganese(ll) [Mn(phen)(S(2)O(3))(H(2)O)(2)] was investigated. Its structure was determined by single crystal X-ray diffraction from 2418 reflections (I > 3 sigma(I)) to a final value of R = 0.047 and Rw = 0.054. Crystal data are as follows : space group P(2) (1); a = 10.356(3), b = 7.097(3), c = 20.316(2) A, beta = 94.29(2) degrees , V = 1489.1(8) , A(3), Z = 2. There are two independent title compounds in the asymetric unit. Each manganese atom has a distorted octahedral Mn(SO)N(2)O(2) geometry with the S and O atoms (from two neighbouring thiosulfate ligands) mutually trans, two N atoms from the 1,10-phenanthroline ligand and two water oxygen. The thiosulfate group behaves as a bridging ligand, connecting, through sulfur and oxygen, Mn atoms related by the binary b translation, thus forming infinite chains running parallel to this axis. Infrared and electronic spectra are reported.

Resistance of Escherichia coli growing as biofilms to disinfectants.

The bactericidal activity of various disinfectants (cationic or amphoteric surfactants, oxidizing agents, phenolic derivatives) was determined against Escherichia coli CIP 54127 obtained by culture on tryptic soy agar (in-suspension or on-germ-carrier test) or in the form of biofilms produced in a continuous culture system. The bacteria tested on germ-carriers or included in biofilms were more resistant than the same strain in suspension. The extent of the reduction in activity depended on the nature of the disinfectant. In the two cases, the greatest reduction was observed with benzalkonium chloride and hexadecyl trimethylammonium bromide, the agents with the lowest hydrophile-lipophile balance. The activity of the oxidizing agents (sodium hypochlorite, peracetic acid/H2O2) and alkyl trimethylammonium derivatives (C12 and C14) was somewhat reduced, while that of the phenolic derivatives (o-cresol, phenol) was either slightly attenuated or unaffected. The reduction in sensitivity was attributed to a reduced accessibility of the bacterial cells to the disinfectants, due to the fact that the former adhered to a support. Furthermore, the interfering action of the substances in contact with the bacteria (milk in the germ-carrier test and exopolymers in the biofilms) could play a role. The reduced sensitivity of the bacteria in the biofilms was not due to any alteration in the metabolic state of the bacteria (mostly in a quiescent state) since this resistance was lost after the mechanical resuspension of the cells before the contact with the disinfectants.

Attachment of Salmonella choleraesuis choleraesuis to beef muscle and adipose tissues.

The attachment of Salmonella choleraesuis subsp. choleraesuis ATCC 15790 to beef muscle and adipose tissues was investigated. S. choleraesuis was found to adhere in higher numbers to muscle than to fat. The charge and the hydrophobicity of the surface of S. choleraesuis were evaluated by measurement of electrophoretic mobility, the contact angle with water, adhesion to hexadecane, and hydrophobic interaction chromatography. The overall negative charge of S. choleraesuis was masked by the high electrolyte concentration in the attachment medium (0.15 M phosphate-buffered saline). This bacterium was shown to possess a hydrophilic surface. Electrostatic interactions do not affect the attachment of S. choleraesuis to both lean and fat tissue, and there was no evidence for a role of hydrophobic interactions. However, the attachment of S. choleraesuis was reduced by 90% after mechanical removal of the flagella or after treatment of the bacteria with specific antiflagella serum. This reduction was attributed to a loss of bacterial mobility leading to a reduction in the number of cells reaching the tissue during the period of contact. Treatment of the tissue with a concentrated suspension of flagella or treatment of the bacteria with antisomatic serum (OMD) did not reduce the attachment of S. choleraesuis to tissues, indicating an absence of specific attachment sites for flagella or antigen O on the beef tissue surface.

Crystallization and preliminary X-ray investigation of barstar, the intracellular inhibitor of barnase.

Crystals of barstar, the intracellular inhibitor of the extracellular ribonuclease produced by Bacillus amyloliquefaciens (barnase), were obtained through vapor phase equilibration using the hanging drop technique. Three crystal forms have been characterized. Forms I and II, crystallized either in potassium phosphate or sodium citrate, are tetragonal; they exhibit a superstructure along the c-axis. Form III crystals, suitable for a high resolution structure determination, were grown from 55-65% ammonium sulfate. This crystal form is hexagonal and diffracts to at least 2 A resolution at a synchrotron radiation source. It belongs to the hexagonal space group P6, with unit cell dimensions a = b = 143.6 A, c = 35.6 A. There are four molecules of barstar in the asymmetric unit. X-ray data have been collected to 2.2 A Bragg spacing. The structure determination is underway in order to analyze conformational changes of barstar upon complexation with barnase.

Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion.

Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC) were found to adhere to the brush border of differentiated human intestinal epithelial Caco-2 cells in culture, whereas Yersinia pseudotuberculosis and Listeria monocytogenes adhered to the periphery of undifferentiated Caco-2 cells. All these enterovirulent strains invaded the Caco-2 cells. Using a heat-killed human Lactobacillus acidophilus (strain LB) which strongly adheres both to undifferentiated and differentiated Caco-2 cells, we have studied inhibition of cell association with and invasion within Caco-2 cells by enterovirulent bacteria. Living and heat-killed Lactobacillus acidophilus strain LB inhibited both cell association and invasion of Caco-2 cells by enterovirulent bacteria in a concentration-dependent manner. The mechanism of inhibition of both adhesion and invasion appears to be due to steric hindrance of human enterocytic pathogen receptors by whole-cell lactobacilli rather than to a specific blockade of receptors.

Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells.

Twenty-five strains of lactobacilli were tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Seven Lactobacillus strains adhered well to the Caco-2 cells, of which three possessed calcium-independent adhesion properties. A high level of calcium-independent adhesion was observed with the human stool isolate Lactobacillus acidophilus strain LB. Scanning electron microscopy revealed that this strain adhered to the apical brush border of the cells. Adhesion increased in parallel with the morphological and functional differentiation of the Caco-2 cells. Two Lactobacillus components were involved in this adhesion. One was protease-resistant and bacterial-surface-associated; the other was heat-stable, extracellular and protease-sensitive.

Heat-killed Lactobacillus acidophilus inhibits adhesion of Escherichia coli B41 to HeLa cells.

Escherichia coli B41 (O101: K99: F41: ST+) adheres to HeLa 229 cells in a diffuse pattern. Heat-killed (100-105 degrees C) Lactobacillus acidophilus (Lactéol strain) was found to inhibit this adhesion in a dose-dependent manner. This inhibitory action was lost after lysis of the L acidophilus, suggesting steric hindrance of E coli adhesion sites rather than competition for a common binding site. A thermostable factor (100-105 degrees C) excreted by L acidophilus into the medium may be required for the adhesion of L acidophilus to HeLa cells, and for the inhibition of adhesion of E coli to these cells.

[Ex-vivo study of the capacity of bacteria of the genus Capnocytophaga to adhere to human buccal epithelial cells]. / Etude ex-vivo de la capacite des bactéries du genre Capnocytophaga à adhérer aux cellules épithéliales buccales humaines.

The bacteria of the genre Capnocytophaga are part of the subdominant flora of the oral cavity. For diverse microorganisms it has been proved that the adhesion constitutes the first step of the colonization of a place leading to the eventual pathology. The adhesion capacity to human epithelial cells (keratinised and non) of eight strains of genus Capnocytophaga has been studied. All strains appear to have a very weak capacity of adhesion. This diministe can be originated from the fact that the host cells have been harvested from healthy subjects.

Activity of capryloyl collagenic acid against bacteria involved in acne.

Synopsis Capryloyl collagenic acid (Lipacide C8Co) has similar bacteriostatic activity in vitro to that of benzoyl peroxide towards the bacteria found in acne lesions (Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes) (MIC between 1 and 4 mg ml(-1) for C8Co, and between 0.5 and 5 mg ml(-1) for benzoyl peroxide). The presence of Emulgine M8 did not affect the bacteriostatic activity of C8Co. A 4% w/v solution of C8Co (incorporating Emulgine M8) fulfilled the criteria for an antiseptic preparation as laid down by the French Pharmacopoeia (10th Edition), and had a spectrum 5 bactericidal activity according to the French Standard AFNOR NF T 72-151. The excellent cutaneous tolerance of capryloyl collagenic acid would indicate that an aqueous solution might be of value for topical treatment of the bacterial component of acne. Résumé Activité antibactérienne de l'acide capryloyl-collagénique vis à vis des bactéries impliquées dans l'etiologie de l'acné L'acide capryloyl-collagénique (Lipacide C8Co) et le peroxyde de benzoyle présentent une activité bactériostatique in-vitroéquivalente vis à vis des espèces bactériennes retrouvées au niveau des lésions acnéiques (Staphylococcus aureus, S. epidermidis et Propionibacterium acnes) (CMI comprise entre 1 et 4 mg ml(-1) pour le lipoaminoacide, et 0,5 et 5 mg ml(-1) pour le peroxyde de benzoyle). La mise en solution aqueuse de l'acide capryloyl-collagénique en présence d'Emulgine M8 ne modifie pas son activité bactériostatique. Une telle solution, à 4% m/V d'acide capryloyl-collagénique et 5% m/V d'Emulgine M8, satisfait à l'essai d'activité des préparations antiseptiques décrit à la Pharmacopée Française (Xème Ed.) (concentration minimale antiseptique: 10% v/V, pour un temps de contact de 5 min à 32 degrees C entre les germes tests et la solution diluée en eau distillée), et posséde une activité bactéricide antiseptique spectre 5 conforme à la norme AFNOR NF T 72-151 (concentration minimale bactéricide: 40% v/V). Cette activité antiseptique est diminuée par l'augmentation du pH, mais demeure significative à pH 5,5, pH moyen du revetement cutané. Compte tenu de la bonne tolérance cutanée de l'acide capryloyl-collagénique, cette solution pourrait constituer une base intéressante pour une preéparation dermique destinée au traitement local de la composante bactérienne de l'acné.

Role of volatile fatty acids in colonization resistance to Clostridium difficile in gnotobiotic mice.

Clostridium difficile is an agent involved in the development of antibiotic-associated pseudomembranous colitis. The purpose of this work was to investigate the role of volatile fatty acids (VFAs) in resistance to colonization by C. difficile by using a gnotobiotic animal model. Accordingly, germfree mice were associated with different hamster flora, and the VFAs in their cecal contents were measured by gas chromatography. The results showed that VFAs were produced mainly by the intestinal flora, especially by the strictly anaerobic bacteria. In these associated mice, the concentrations of acetic, propionic, and butyric acids were higher than those of other acids, but at pH 6.8 the MICs of these three acids in vitro for C. difficile were more than 200 mu eq/ml. In gnotobiotic mice monoassociated with C. difficile and in the isolated ceca of these mice, VFAs did not inhibit the growth of C. difficile. In gnotobiotic mice which were diassociated with C. difficile and C. butyricum and given drinking water with a lactose concentration of 20%, the cecal contents included about the same amount of butyric acid as did those of the monoassociated mice, although the population of C. difficile remained the same. Therefore, it is suggested that VFAs alone cannot inhibit intestinal colonization by C. difficile and that, consequently, other inhibitory mechanisms are also present.

[Transfer of the cecal flora of the hamster to the germfree C3H mouse: use of this model to study the flora of the anti-Clostridium difficile barrier]. / Transfert de la flore caecale du hamster à la souris C3H axénique: utilisation de ce modèle pour étudier la flore de barrière anti-Clostridium difficile.

The purpose of this work was the research and development of an experimental model to study anti-Clostridium difficile caecal microflora in the hamster. First the existence of this "barrier" was verified in conventional hamsters. Then, the caecal flora from these animals was orally transferred to C3H germfree mice. The barrier effect was maintained in the axenic mice. The comparative bacteriological analysis of hamster and mouse feces did not reveal important variations in the dominant anaerobic flora (P less than 0.01). After treatment with erythromycin, the barrier effect was maintained and while the disappearance of Escherichia coli was observed, the dominant anaerobic flora persisted. After dilution (10(-2] and subsequent heating (70 degrees C, 10 min) of caecal contents, the inhibitory activity against C. difficile was maintained although the number of aerobic and aerotolerant bacteria was reduced. The isolation from caecal microflora of anaerobic strains implicated in the resistance to colonization is presently underway in Freter anaerobic chambers.

[Evaluation of an experimental animal model allowing the study of the cecal microflora in the hamster, antagonistic to clostridium difficile]. / Mise au point d'un modèle expérimental animal permettant l'etude de la microflore coecale du hamster, antagoniste de Clostridium difficile.

The purpose of this study was the development and evaluation of an experimental model allowing the investigation of hamster anti-Clostridium difficile coecal microflora. The existence of this "barrier" was verified in conventional hamsters. Such hamster coecal flora was then orally transferred to C3H germ-free mice. In such animals, the "barrier effect" was maintained. After treatment with erythromycin, the colonization resistance was always maintained; despite two subsequent processes, dilutions of coecal contents (10(-2] and subsequent heating of this fluid (70 degrees C, 10 min), the inhibitory activity against C. difficile was partially maintained (10(4) UFC/g faeces). The isolation of anaerobic strains implicated in colonization resistance will next be carried out in an anaerobic chamber using this microflora.

[Effect of the administration of killed Lactobacillus acidophilus on the survival of suckling mice infected with a strain of enterotoxigenic Escherichia coli]. / Influence de l'administration de Lactobacillus acidophilus tués sur la survie de souriceaux infectés avec une souche de Escherichia coli entérotoxinogène.

After oral administration of 10(2) to 10(5) CFU of Escherichia coli B41 (0101:K 99+:ST+) to 24-48 h old suckling mice (Swiss OF1), a 80 to 100% mortality rate is observed within three days. We compared the effect of the oral treatment with a lyophilized preparation of heat-killed Lactobacillus acidophilus and with sterile water on the mortality rate of newborn mice. In six out of seven assays, the heat-killed L. acidophilus administration extended survival of infected suckling mice (P ranging from 0.019 to less than 0.001). In another test, result was contradictory. A global analysis indicated that the two treatments were statistically different (P less than 0.001). Lactic acid was unable to induce protection.

[Effect of yogurt ingestion on the intestinal lactase activity in axenic or holoxenic mice]. / Influence de l'ingestion de yogourt sur l'activité lactasique intestinale chez des souris axéniques ou holoxéniques.

Two sets of mice just weaned (germ-free or conventional) were fed ad libitum with three different diets: a 5% lactose solution, yogurt at a 3/5 dilution in this lactose solution, and heated yogurt at the same dilution in the same solution. Total lactase activity (TA) and specific lactase activity (SA) of the small intestine were measured after 14, 28 and 42 days of these diets. The results led to the following conclusions. 1) TA and SA were always significantly greater in germ-free and gnotobiotic animals than in conventional ones, whatever the diets and age of the animals. 2) TA and SA were significantly greater at the three times of measurement in the gnotobiotic and conventional animals fed with unheated yogurt than in the animals fed with heated yogurt. The 5% lactose solution gave intermediate results. 3) In conventional and gnotobiotic animals fed with yogurt, TA increased with time compared to the initial value at weaning. With the two other diets, TA remained constant or decreased. With yogurt, SA varied only weakly with time compared to weaning values, when it decreased in greater proportions with the two other diets.

[Biochemical characterization of Haemophilus strains with a standardized micromethod (author's transl)]. / Détermination des carcatères biochimiques des germes du genre Haemophilus par l'utilisation d'une microméthode normalisée.

Using a standardized micromethod, the authors aimed at the study of biochemical characterization of 51 Haemophilus strains (influenzae and parainfluenzae). The statistical analysis of the results allowed them to definite a theoretical average outline corresponding to three groups of bacteria :-- group of Haemophilus influenzae, -- group of Haemophilus parainfluenzae, -- group of different Haemophilus. The authors propose some modifications in order to make this identification specifically applicable to diagnose species and biotypes of genus Haemophilus.