Role of FliC and FliD flagellar proteins of Clostridium difficile in adherence and gut colonization.

In vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins of Clostridium difficile were analyzed. FliC, FliD, and crude flagella adhered in vitro to axenic mouse cecal mucus. Radiolabeled cultured cells bound to a high degree to FliD and weakly to flagella deposited on a membrane. The tissue association in the mouse cecum of a nonflagellated strain was 10-fold lower than that of a flagellated strain belonging to the same serogroup, confirming the role of flagella in adherence.

Contribution of capillary zone electrophoresis to the analysis of cecal mucins.

The ability of capillary electrophoresis to perform the separation of mucin glycoforms has been investigated. Adsorption of mucins to the capillary was observed in most cases, leading to unreproducible results. This was due in part to the characteristic structure of mucin (highly charged, large size) and also to its poor solubility. Various buffers were therefore investigated; it was found that a zwitterionic electrolyte, such as a (3-[cyclohexylamino]-1-propanesulfonic acid) (CAPS) buffer, pH 8.8, greatly improved the separation. Using this buffer, mucin was resolved into five main fractions. The use of several additives, such as cationic molecules (1,4-diaminobutane [DAB]) or hydrophilic polymers (hydroxypropylmethylcellulose [HPMC], polyethylene glycol [PEG]) was also investigated. PEG and HPMC did not affect the separation and the electroosmotic flow (EOF) in the same manner. The favorable effect of the addition of PEG was clearly demonstrated and it was postulated that some interaction of this polymer with the mucins occurred. Finally, the application of the method to the comparison of glycoform patterns of mouse cecal mucins showed a marked difference for mucins derived from two sources: germ-free and gnotobiotic mice. These results indicate that mucins from gnotobiotic mice have been degraded due to the glycosidic activity of the bacterial strains present in these mice.

Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.

Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment.

Identification of a Clostridium cocleatum strain involved in an anti-Clostridium difficile barrier effect and determination of its mucin-degrading enzymes.

We isolated Gram-positive circular bacterium HB1 from intestinal microflora showing resistance to colonization by Clostridium difficile in mice (Su et. al., 1986a,b). We studied its enzymatic capacity to degrade mucin the first potential barrier to implantation of strains in the intestine. Its biochemical characteristics, terminal metabolites and the electrophoretic profiles of proteins and DNA-DNA homology indicated that it was a strain of Clostridium cocleatum. This strain displayed numerous glucosidase activities which were assumed to play a role in the degradation of mucin oligosaccharide chains in the digestive tract. These enzymes included alpha- and beta-galactosidases, beta-glucosidase, beta-N-acetylglucosaminidase, sialidase and alpha-N-acetylgalactosaminidase.

In vitro antimicrobial activity of 18 phenothiazine derivatives: structure-activity relationship.

The antimicrobial activity of 18 phenothiazine derivatives has been evaluated against aerobic and anaerobic bacteria. The mean of the MIC reveals five active drugs against the bacterial strains tested: thioridazine, chlorpromazine, trifluoperazine, fluphenazine and triflupromazine. The other derivatives are less active and some show no antimicrobial activity. It seems that in vitro antibacterial activity alone is insufficient to explain certain intestinal diseases linked to the use of these molecules in psychiatry.

[Bacterial interference with skin colonization in germfree hairless mice]. / Interferences bacteriennes au niveau cutane chez la souris "hairless" dixénique.

Bacterial colonization of the digestive tract and the skin was studied over a 3-week period in a group of 10 germfree HRS mice using Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa. Sequential utilization of two strains allowed us to carry out six assays and to show the presence of interference phenomena during colonization of the skin. When P. aeruginosa was given after challenge with S. aureus or S. epidermidis, it did not colonize the skin. If the first challenge was done with P. aeruginosa, this bacteria was eliminated within 10 days by S. aureus and S. epidermidis on the skin, but it succeeded in colonizing the digestive tract. When the first challenge was done with S. aureus, colonization of the skin and the digestive tract with S. epidermidis was prevented, whereas these two species were found in association when S. aureus was given in second place. None of the in vitro assays (mixed culture, bacteriocin production, adherence inhibition, antimicrobial activity) could explain the in vivo observations.