Marie-Claude Barc

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

ABSTRACT In vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins ofClostridium 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.

GroEL (Hsp60) of Clostridium difficile is involved in cell adherence

Previous results have demonstrated that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses; this study focussed on whether the GroEL heat shock protein is implicated in this process. The 1940 bp groESL operon of C. difficile was isolated by PCR. The 1623 bp groEL gene is highly conserved between various C. difficile isolates as determined by RFLP-PCR and DNA sequencing, and the operon is present in one copy on the bacterial chromosome. The 58 kDa GroEL protein was expressed in Escherichia coli in fusion with glutathione S:-transferase and the fusion protein was purified from IPTG-induced bacterial lysates by affinity chromatography on glutathione-Sepharose. A polyclonal, monospecific antiserum was obtained for GroEL which established by immunoelectron microscopy, indirect immunofluorescence and immunoblot analysis that GroEL is released extracellularly after heat shock and can be surface associated. Cell fractionation experiments suggest that GroEL is predominantly cytoplasmic and membrane bound. GroEL-specific antibodies as well as the purified protein partially inhibited C. difficile cell attachment and expression of the protein was induced by cell contact, suggesting a role for GroEL in cell adherence.

Clostridium difficile genotyping based on slpA variable region in S-layer gene sequence: an alternative to serotyping.

ABSTRACT Recent investigations of Clostridium difficile cell wall components have revealed the presence of an S-layer encoded by the slpA gene. The aim of this study was to determine whether slpA genotyping can be used as an alternative to serotyping. The variable regions of slpA were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly. Amplified products were analyzed after restriction enzyme digestion and DNA sequencing. The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups. These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the slpA variable region, could constitute an alternative typing method for determining C. difficile serotypes.

A Clostridium difficile gene encoding flagellin

Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella. The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined. Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins. Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains. The two gene sequences exhibited 91% inter-strain identity. The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE. The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable. A second ORF is present downstream of fliC displaying homology to glycosyltransferases. The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific antiserum was obtained. Flagella of C. difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells. PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism.

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.

Clostridium difficile in Ground Meat, France

To the Editor: Clostridium difficile is a toxigenic enteropathogen responsible for 15%–20% of antimicrobial drug–associated diarrhea and for almost all cases of pseudomembranous colitis. Two protein toxins (TcdA and TcdB) play a major role in the pathogenesis of infections. C. difficile is also recognized as a cause of disease in several animal species, which could be potential reservoirs (1). In the past few years, the presence of C. difficile in raw diets for dogs and cats and in retail meat sold for human consumption has been reported in the United States and Canada at rates from 6% to 42% (2–5). To determine C. difficile contamination of meat in France, we evaluated 105 packages of ground beef (vacuum packed or not), 59 pork sausages, and 12 packages of feline raw diet meat purchased from 20 urban and suburban Paris retail stores and supermarkets during September 2007–July 2008. C. difficile spores or vegetative forms in samples were found as described by Rodriguez-Palacios et al. (4). Briefly, 5 g of each samplewas cultured in 100 mL of prereduced brain–heart infusion (BHI) broth supplemented with cefoxitin (10 µg/mL), cycloserine (250 µg/mL), and taurocholate (0.1%). After the samples were incubated under anaerobic conditions at 37°C for 72 h, subculturing with and without alcohol shock for spore selection was performed. The BHI broth culture was treated with 2 mL of absolute ethanol (1:1 vol/vol) for 30 min and centrifuged at 3,800 × g for 10 min, and the pellet was resuspended in 200 µL of prereduced BHI broth. Serial dilutions of the BHI broth and the pellet were injected onto Columbia cysteine agar supplemented with cefoxitin-cycloserine, taurocholate, and 5% horse blood and incubated anaerobically for 48 h at 37°C. C. difficile colonies were identified classically, and susceptibilities to moxifloxacin, teicoplanin, vancomycin, metronidazole, linezolid, levofloxacin, telithromycin, erythromycin, and lincomycin were determined by the agar disk-diffusion methods described by the French Society for Microbiology (www.sfm.asso.fr). PCR amplifications of a species-specific internal fragment of the triose phosphate isomerase (tpi) gene, an internal fragment of the toxin B (tcdB) gene, and the 3′ region of the toxin A (tcdA) gene were performed as described by Lemee et al. (6). Strains were characterized by toxinotyping according to Rupnik et al. (7) and PCR-ribotyping as described by Bidet et al. (8). The detection threshold of the enrichment method was established by spiking known uninfected samples (ground beef, pork sausage, and feline raw diets) with vegetative cells and spores of C. difficile (VPI 10463 strain). For ground beef samples, the detection thresholds for vegetative forms and spores were 2 CFU/5 g and 4.5 CFU/5 g of meat, respectively. For pork sausages, the detection thresholds were 14 CFU/5 g and 38 CFU/5 g of sample after 72 h, for vegetative forms and spores, respectively. For feline raw diets, the detection threshold of spores was 2 CFU/5 g of sample. In addition, toxin B was detected in the culture supernatants by the cytotoxicity assay onto MRC-5 cells. Toxin detection showed 100% agreement with the culture method. C. difficile was not detected in pork sausages or in commercial feline raw diets. C. difficile was isolated from 2 (1.9%) of 105 ground beef, but only from those packages that were vacuum packed. The anaerobic atmosphere of vacuum packaging could facilitate the survival of C. difficile and the germination of spores. These 2 isolates were fully susceptible to moxifloxacin, teicoplanin, vancomycin, metronidazole, and linezolid but resistant to levofloxacin, telithromycin, erythromycin, and lincomycin. They harbored genes encoding for Tpi protein and for TcdA and TcdB. The 2 strains belonged to the toxinotype 0 and PCR-ribotype 012. Toxinotype 0 was already identified in meat samples in Canada (4). PCR-ribotype 012 belongs to the 10 ribotypes most frequently isolated from humans (9). The prevalence of C. difficile in ground meat in France is low compared with the prevalence reported by other countries. In Canada, Rodriguez-Palacios et al. (4) studied 60 beef samples and found the prevalence of C. difficile to be 20%. These same authors, by using a broader sampling scheme (214 meat samples), isolated C. difficile from 6% of the samples (5). Also in Canada, Weese et al. (10) reported that 12% of ground beef and ground pork samples were contaminated. In the United States, C. difficile was isolated from 42% of meat samples (beef, pork, and turkey products) (3). For a better understanding of the sources of C. difficile in France, it would be interesting to determine its prevalencein different animal fecal samples and the toxinotypes associated with animals. The low prevalence in retail meat in France could result from hazard analysis critical control point principles and microbiologic quality controls implemented throughout the food production chain, which help reduce the spread of C. difficile and minimize the risk for infection for the consumer.