[Saccharomyces boulardii modulates dendritic cell properties and intestinal microbiota disruption after antibiotic treatment]. / Saccharomyces boulardii module les propriétés descellules dendritiques et le déséquilibre du microbioteintestinal après un traitement antibiotique.

Saccharomyces boulardii is a non-pathogenic yeast with biotherapeutic properties that has been used successfully to prevent and to treat various infectious and antibiotic-associated diarrheas. The intestinal microbiota is responsible for colonization resistance and immune response to pathogens but can be disrupted by antibiotics and lose its barrier effect. Dendritic cells (DCs) are professional antigen-presenting cells of the immune system with the ability to initiate a primary immune response or immune tolerance. In a human microbiota-associated mouse model, we evaluated the influence of S. boulardii on the composition of the microbiota and on the properties of dendritic cells in normal homeostatic conditions and after antibiotic-induced stress. The DCs were derived from splenic precursors. Membrane antigen expression and phagocytosis of FITC-latex beads by DCs were evaluated by flow cytometry. The molecular analysis of the microbiota was performed with fluorescence in situ hybridization (FISH) combined with flow cytometry or confocal microscopy using group specific 16S rRNA targeted probes. This evaluation was conducted during and after a 7-day oral treatment with amoxicillin-clavulanic acid alone and in combination with the administration of the yeast. The antibiotic treatment increased the phagocytic activity of DCs. Their antigen presenting function (MHC class II antigen and CD 86 costimulatory molecule membrane expression) was up-regulated. This reflects a functional activation of DCs. In the presence of S. boulardii, the modification of membrane antigen expression was down regulated. To correlate these modifications to the microbiota disruption, we analyzed in parallel the composition of the intestinal microbiota. As previously shown, the amoxicillin-clavulanic acid treatment, both alone and with S. boulardii, did not quantitatively alter the total microbiota. In contrast, after one day of the antibiotic treatment the Clostridium coccoides group decreased dramatically in the two groups of mice treated with the antibiotic. The level then increased regularly, and at days 17, 22 and 24 it increased faster (P < 0.05) in the AB+ Sb group than in the AB group, reaching the initial level at day 29. The Bacteroides group in the two groups of mice increased during the antibiotic treatment and decreased after the antibiotic was stopped, reaching the initial level. The rate of decrease was faster for the AB+ Sb group than for the AB group, with a significant difference (P < 0.05) at days 17 and 22. During antibiotic treatment, the Enterobacteriaceae group became detectable and its level increased in both groups of mice. After discontinuation of the antibiotic, its level decreased to become undetectable at day 29, without significant difference between the two groups. These results showed that S. boulardii treatment tends to restore the balance of the dominant anaerobic microbiota more rapidly in human microbiota associated-mice treated with amoxicillin-clavulanic acid; the results also suggest that the yeast has a role in modulating the specific immune response to microbial associated-molecular patterns. This may explain, at least in part, the beneficial effects of S. boulardii in preventing antibiotic-associated diarrhea. This also suggests that the yeast plays a role in maintaining intestinal homeostasis.

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.

Molecular characterization of fliD gene encoding flagellar cap and its expression among Clostridium difficile isolates from different serogroups.

The fliD gene encoding the flagellar cap protein (FliD) of Clostridium difficile was studied in 46 isolates belonging to serogroups A, B, C, D, F, G, H, I, K, X, and S3, including 30 flagellated strains and 16 nonflagellated strains. In all but three isolates, amplification by PCR and reverse transcription-PCR demonstrated that the fliD gene is present and transcribed in both flagellated and nonflagellated strains. PCR-restriction fragment length polymorphism (RFLP) analysis of amplified fliD gene products revealed interstrain homogeneity, with one of two major patterns (a and b) found in all but one of the strains, which had pattern c. A polyclonal monospecific antiserum raised to the recombinant FliD protein reacted in immunoblots with crude flagellar preparations from 28 of 30 flagellated strains but did not recognize FliD from nonflagellated strains. The fliD genes from five strains representative of the three different RFLP groups were sequenced, and sequencing revealed 100% identity between the strains with the same pattern and 88% identity among strains with different patterns. Our results show that even though FliD is a structure exposed to the outer environment, the flagellar cap protein is very well conserved, and this high degree of conservation suggests that it has a very specific function in attachment to cell or mucus receptors.

Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups.

Phenotypic and genotypic diversity of the flagellin gene (fliC) of Clostridium difficile was studied in 47 isolates from various origins belonging to the serogroups A, B, C, D, F, G, H, I, K, X, and S3. Electron microscopy revealed 17 nonflagellated strains and 30 flagellated strains. PCR and reverse transcription-PCR demonstrated that the flagellin gene was present in all strains and that the fliC gene was expressed in both flagellated and nonflagellated strains. Southern blotting showed the presence of only one copy of the gene and three different hybridization patterns. DNA sequence analysis of fliC from the strains belonging to serogroups C, D, and X, representative of each profile, disclosed great variability in the central domain, whereas the N- and C-terminal domains were conserved. The variability of the flagellin gene fliC was further studied in the isolates by PCR-restriction fragment length polymorphism (RFLP) analysis. Nine different RFLP groups were identified (I to IX), among which three (I, VII, and VIII) corresponded to numerous serogroups whereas the six others (II, III, IV, V, VI, and IX) belonged to a single serogroup. Flagellin gene RFLP analysis could constitute an additional typing method employable in conjunction with other typing methods currently available.

Clostridium difficile cell attachment is modified by environmental factors.

Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa.

Protease activity of Clostridium difficile strains.

The production of proteolytic enzymes by 10 Clostridium difficile isolates of varying toxigenicity and clinical origin was studied to determine if all isolates secreted proteases. Different protease substrates were studied: gelatin, collagen, phenylazobenzyloxycarbonyl-leucyl-glycyl-L-prolyl-D-arginine (Pz-peptide), casein, azocasein, and azocoll. All isolates degraded gelatin, collagen, and azocoll. The supernatants of all isolates contained an enzyme capable of attacking gelatin incorporated in a polyacrylamide gel (zymograms) and forming two closely spaced lytic bands with an estimated molecular mass of 35-40 kDa. Polyclonal antibodies, produced against the C. difficile gelatinase, revealed in Western blots a 35-kDa protein in the culture supernatants of all C. difficile isolates. In the same manner, Clostridium perfringens collagenase polyclonal antibodies detected a 120-kDa protein in the culture supernatants of all isolates; this suggests that at least two proteases may exist in C. difficile. The protease activities of the 10 strains examined did not seem strikingly different quantitatively but were in general weak and their role in pathogenicity is suspect.

Randomly amplified polymorphic DNA analysis provides rapid differentiation of methicillin-resistant coagulase-negative staphylococcus bacteremia isolates in pediatric hospital.

Coagulase-negative staphylococci (CoNS) are now recognized as the most common cause of nosocomial bacteremia in pediatric patients. Randomly amplified polymorphic DNA analysis was used to study the relationships among 12 isolates of CoNS obtained from eight patients with catheter-related bacteremia in two distinct wards of our hospital and 6 epidemiologically unrelated strains. With this method, we were able to discriminate isolates that otherwise were indistinguishable by conventional criteria such as biochemical typing and antibiotic susceptibility patterns. Our results indicate that there were episodes of cross-infections among four patients in one ward but independent infectious episodes among four patients in the other ward. Randomly amplified polymorphic DNA analysis is a rapid method which seems particularly well suited to the epidemiological study of CoNS isolates.

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 and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture.

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was conducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhesion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27 kDa proteins. Indeed, adhesion to Caco-2 cell monolayers of C. difficile strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficile may utilize blood components as adhesins to adhere to human intestinal cultured cells.

Effects of antibiotics and other drugs on toxin production in Clostridium difficile in vitro and in vivo.

In an attempt to understand more completely why patients treated with phenothiazines (chlorpromazine and cyamemazine), methotrexate, and certain antibiotics such as clindamycin have an increased risk of developing pseudomembranous colitis, the production of toxins A and B by Clostridium difficile in the presence of these drugs was measured in vitro as well as in vivo by using axenic mice. None of the drugs tested increased the production of toxins either in vitro or in vivo.

In vivo standardization of cutaneous bactericidal activity of antiseptics by using monoxenic hairless mice.

This study was designed to investigate the bactericidal activities of antiseptics on the cutaneous flora of hairless mice monoxenic to Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa in vivo. A standardized method for testing such antiseptics to compare their bactericidal effectiveness in humans in described. Seven antiseptics belonging to seven different chemical groups (iodine derivatives, alcohols, mercury compounds, quaternary ammonium salts, biguanides, phenols, and carbanilides) were used as recommended by the manufacturers (conditions of contact or prolonged contact time followed by washing with distilled water). Germfree hairless mice were infected with various bacterial strains by gastric intubation, producing levels of about 10(3) CFU/cm2 of skin. The antiseptic under test was placed on the right or left side of the ventral region. The contralateral side served as a control. In order to standardize the method, a number of crucial parameters were carefully controlled: the amount of antiseptic applied, the area of skin treated, the duration of treatment, and the washing procedure. Skin samples were obtained by cutaneous biopsy, which effectively removed all the bacteria along with the sample. The bacterial populations were counted before and after application of the antiseptic. Reductions of between 0.5 and 1.9 log units were obtained; these are comparable to those observed in humans. The standardization of our procedure and the use of animals with a strictly controlled flora eliminated much of the variability and sources of error inherent in human studies. This model could be of value for the study of resistant bacterial strains responsible for nosocomial infections and for investigations of damaged skin.

[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.

[Digestive and cutaneous colonization of Staphylococcus aureus, Pseudomonas aeruginosa and Corynebacterium xerosis in germ-free hairless mice]. / Implantation digestive et cutanée de Staphylococcus aureus, Pseudomonas aeruginosa et Corynebacterium xerosis chez la souris hairless axénique.

Regardless of the method of germ-free HRS mouse contamination, the kinetics of Staphylococcus aureus and Pseudomonas aeruginosa cutaneous colonization was parallel to the kinetics of intestinal colonization; Corynebacterium xerosis, however, was unable to colonize the skin or the gastrointestinal tract. There was little variability in intestinal and cutaneous colonization; this was quite different from observations in man and hairless holoxenic mice. It is possible to use these experimental models to standardize the measurement of antiseptic bactericidal activity in vivo.

[Experimental models for the study of antiseptics]. / Modèles expérimentaux d'étude des antiseptiques.

Determination of the bactericidal concentration of antiseptics in vitro is now standardized, allowing interesting comparisons. Conversely, measurement of this activity in vivo in man is difficult since results are dependent upon skin sampling techniques and variations in bacterial growth from individual to individual and from one site to the next. Skin sampling can be standardized by using the hologenic Hairless mouse as an experimental model (skin needle biopsy and crushing). However, variations from one animal to the next remain a problem, even though results are very similar to those found in man. We therefore used monoxenic Hairless mice contaminated with S. aureus, S. epidermidis and P. aeruginosa. All experiments on these animals demonstrated consistency of bacterial cutaneous and digestive growth over time and smallness of variations in bacterial flora from animal to animal. We advocate use of this second model for standardization of evaluation of bactericidal activity of antiseptics.

[Digestive and cutaneous colonization of Staphylococcus epidermidis in hairless axenic mice]. / Implantation digestive et cutanée de Staphylococcus epidermidis chez la souris axénique "hairless".

In order to determine the cutaneous and digestive colonization of S. epidermidis in germ-free HRS mice, several groups of animals were contaminated with this strain according to different methods. The bacterial cutaneous enumerations were performed after preliminary crushing of the cutaneous biopsy. In this study, whatever the method of contamination of germ-free HRS mice, the kinetics of S. epidermidis cutaneous colonization was parallel to the kinetics of intestinal colonization. The isolation of bacteria on skin was possible only when microorganisms were eliminated in faeces (6 h after contamination). During a single experiment, there was only slight variability in colonization, whether intestinal or cutaneous. Some differences could be found from one experiment to another, but all animals in the same trial showed the same cutaneous bacterial count. This experimental model is characterized by an interrelation between the intestinal and cutaneous ecosystems.

[Evaluation of bactericidal activity of antiseptics on the cutaneous flora of holoxenic hairless mouse]. / Evaluation de l'activité bactéricide des antiseptiques sur la flore cutanée de la souris hairless holoxénique.

Holoxenic Hairless mice were used in an experimental model to study the in vivo bactericidal activity of several antiseptics. In these experiments skin was sampled by biopsy; after contact between antiseptic and skin, bactericidal activity was neutralized. The results obtained with several products (polyvinylpyrrolidone iodine, alcoholic iodine, ethanol, chlorhexidin, benzalkonium chloride, mercurothiolic acid, trichlorocarbanilid and hexachlorophen) were quite similar to those obtained on human skin. The differences must be related to the composition of the Hairless mouse skin flora which contains Staphylococcus xylosus, Micrococcus sp. and Streptococcus faecalis, but does not contain Corynebacterium sp. or Propionibacterium sp.