Pierre Bourlioux

Characterization of a cell surface protein of Clostridium difficile with adhesive properties.

ABSTRACT Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60°C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The cwp66 gene, encoding a cell surface-associated protein of C. difficile 79-685, was isolated by immunoscreening of a C. difficile gene library with polyclonal antibodies against C. difficile heated at 60°C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of Bacillus subtilis. A survey of 36 strains ofC. difficile representing 11 serogroups showed that the 3′ portion of the cwp66 gene is variable; this was confirmed by sequencing of cwp66 from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase inEscherichia coli and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence ofC. difficile to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia.

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 con‐ducted. 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 adhe‐sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco‐2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat‐shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.

Molecular Characterization of fliD Gene Encoding Flagellar Cap and Its Expression among Clostridium difficile Isolates from Different Serogroups

ABSTRACT 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 amplifiedfliD 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 fliDgenes 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.

Mise au pointPourquoi la flore intestinale a-t-elle vocation à devenir médicament ?Why could gut microbiota become a medication?

The gut microbiota (or gut flora) is a set of bacteria living in symbiosis with the host. Strictly associated with the intestinal tract and interacting with it, the gut microbiota is not a tissue nor an organ, but a supra-organism. A disruption of dialogue between bacteria and human cells is a risk factor or a possible cause of various diseases. The restoration of this dialogue, thanks to the transfer of the gut microbiota of a healthy individual to a patient whose balance of gut flora has been broken, is a new therapeutic approach. If its exact effect still eludes scientific understanding, its clinical benefit is well established for an indication, and is recently being tested for many others. The proven contribution of gut microbiota in the human physiological balance calls for intensifying research throughout the world about the state of knowledge and technologies, as well as on the legal and ethical dimension of fecal microbiota transfer. This didactic paper updates the questions in relation with this therapeutic act.

Le transfert de microbiote fécal lors d’infections récidivantes à Clostridium difficile. Cadre et aspects pharmacotechniques

The fecal microbiota transplantation consists in introducing a preparation constituted by a dilution of stools of a healthy donor in the digestive tract of a patient recipient, to restore his intestinal physiological balance. This therapeutic approach was the subject of numerous studies showing its efficiency in the treatment of the recurrent infections with Clostridium difficile. The fecal microbiota transplantation has now a high level of clinical evidence, which explains that it appears in various international recommendations. In France, the fecal microbiota transplantation responds to the definition of a medication and can be executed as a pharmaceutical preparation or as an experimental drug for clinical trials under the responsibility of a hospital pharmacy. The objective of this paper is to propose a definition of a framework and to describe the methods of preparation of the fecal microbiota transplantation in the treatment of the recurrent infections with C.xa0difficile and the interactions to consider for hospital pharmacies that do not have technical means to operate this technique.

Bacteriological validation of a new apparatus for disinfection of hospital waste at the point of disposal.

OBJECTIVESnTo evaluate the ability of a new apparatus (Dipsys 25, Société SGN, Bagnols sur Cèze, France) to disinfect biomedical waste, including both potentially infectious agents and the normal saprophytic flora of the waste.nnnMETHODSnDisinfection was assessed using standard methods (reference strains were fixed on reference carriers according to the French AFNOR methods) and nonstandard assays. Assays in conditions of hospital use, evaluations of bacterial survival during storage, sporicidal effect, and spore survival during storage were performed in parallel. Finally, bactericidal effect in extreme conditions (association of high contamination and high bacterial protection conditions) was tested with normal fecal flora. Bacterial counts were performed after treatment by the apparatus and without treatment (controls). All tests were carried out in triplicate.nnnRESULTSnIn all treated carriers, a bacterial population decrease of at least 5 log10 was obtained. Assays performed in hospital-use conditions did not show any bacterial growth. Concerning the evaluation of sporicidal effect and spore revival during conservation, a minimum reduction of 5 log10 was observed in all assays performed, without survival. Finally, concerning assays in extreme conditions, the decrease of bacterial population was between 5 log10 and 10 log10 for vegetative anaerobes of normal fecal flora.nnnCONCLUSIONnUnder our study conditions, the study apparatus reduced the tested microbial populations by a minimal factor of 5 log10. The main advantage of the apparatus is the opportunity to treat contaminated waste inside hospital wards, at the point of initial collection, without pulverization, by nonspecialized staff.