1997: Accreditation for research direction (Habilitation à diriger les recherches, HDR)
1994/1998: Ph.D. in Microbiology, Paris VI University, Paris Subject: Streptococcus pneumoniae resistance to fluoroquinolones. Mentor: Pr. L. Gutmann LRMA (Laboratory of molecular research on antibiotics), directed by Dr. E. Collazt.
Abstract: We have designed an oral vaccine against Clostridium difficile infection. The virulent factor Cwp84, that is a cystein protease highly immunogenic in patients with C. difficile-associated disease, was entrapped within pectin beads. Beads encapsulating Cwp84 were shown to be stable in the simulated intestinal medium and to release the cystein protease once in the simulated colonic medium. Three groups of hamsters were immunized, the first receiving pectin beads encapsulating Cwp84, the second unloaded beads and the third one free Cwp84. After three immunizations by the intragastric route, all groups received clindamycine. Post-challenge survival with a strain of C. difficile showed that 2days after infection, all hamsters treated with unloaded beads and all hamsters treated with free Cwp84 have deceased after 7days, whereas about 40% of hamsters administered with Cwp84-loaded beads survived 10days after challenge, proving that oral vaccination provides partial protection. These first data obtained with an oral vaccine against C. difficile appear promising for preventing this infection.
Abstract: The Sirscan2000automatic MIC determination (SIR-MD) system is a new system for MIC determination based on the automatic detection of growth of bacteria spotted onto agar medium using a camera scan. To evaluate its accuracy, 3608 Streptococcus pneumoniae clinical isolates yielding 18,165 MICs were tested in parallel with the SIR-MD and a standard interpretive antibiogram procedure. The overall percent agreement between the 2 methods within 1 log(2) dilution was 86.9%. After exclusion of the 11.8% noninterpretable results, errors in the deduction of susceptibilities were very major in 0.03%, major in 0.2%, and minor in 1.3%.
Abstract: Strains of Clostridium difficile produce a number of surface-localized proteins, including the S-layer proteins (SLPs) and other proteins that have suspected roles in pathogenesis. During the Third International C. difficile Symposium (Bled, Slovenia, September 2010) discussions were held on standardization of nomenclature. Gene designations were proposed for the large family of cell wall proteins that are paralogues of the SLP and contain putative cell wall binding motifs. This paper summarizes the agreed nomenclature, which we hope will be used by research groups currently active in the field.
Abstract: Clostridium difficile is a pathogen responsible for diarrhoea and colitis, particularly after antibiotic treatment. We evaluated the C. difficile protease Cwp84, found to be associated with the S-layer proteins, as a vaccine antigen to limit the C. difficile intestinal colonization and therefore the development of the infection in a clindamycin-treated hamster model. First, we evaluated the immune response and the animal protection against death induced by several immunization routes: rectal, intragastric and subcutaneous. Antibody production was variable according to the immunization routes. In addition, serum Cwp84 antibody titres did not always correlate with animal protection after challenge with a toxigenic C. difficile strain. The best survival rate was observed with the rectal route of immunization. Then, in a second assay, we selected this immunization route to perform a larger immunization assay including a Cwp84 immunized group and a control group. Clostridium difficile intestinal colonization and survival rate, as well as the immune response were examined. Clostridium difficile hamster challenge resulted in a 26% weaker and slower C. difficile intestinal colonization in the immunized group. Furthermore, hamster survival in the Cwp84 immunized group was 33% greater than that of the control group, with a significant statistical difference.
Abstract: Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ∼80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein. Moreover, we showed that the complete maturation of the recombinant protein Cwp84(30-803) is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp84(92-518) (47 kDa). In contrast, a fragment including the propeptide (Cwp84(30-518)) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities.
Abstract: Clostridium difficile, a leading cause of hospital-acquired infection, possesses a dense surface layer (S-layer) that mediates host-pathogen interactions. The key structural components of the S-layer result from proteolytic cleavage of a precursor protein, SlpA, into high- and low-molecular-weight components. Here we report the discovery and optimization of the first inhibitors of this process in live bacteria and their application for probing S-layer processing. We also describe the design and in vivo application of activity-based probes that identify the protein Cwp84 as the cysteine protease that mediates SlpA cleavage. This work provides novel chemical tools for the analysis of S-layer biogenesis and for the potential identification of novel drug targets within clostridia and related bacterial pathogens.
Abstract: The disease spectrum caused by Clostridium difficile infection ranges from antibiotic-associated diarrhoea to life-threatening clinical manifestations such as pseudomembranous colitis. C. difficile infection is precipitated by antimicrobial therapy that causes a disruption of the normal colonic microbiota, predisposing to C. difficile intestinal colonisation. The pathogenicity of C. difficile is mediated by two exotoxins, TcdA and TcdB, both of which damage the human colonic mucosa and are potent cytotoxic enzymes. C. difficile must first be implanted in the gut and attach to epithelial cells, which are protected by a layer of dense mucus. Confirmed and putative accessory virulence factors that could play a role in adherence and intestinal colonisation have been identified and include proteolytic enzymes and adhesins. Recently, the epidemiology of C. difficile infection has radically changed and an increased incidence is associated with outbreaks in North America and Europe. Several reports suggest that disease severity is increasing to include sepsis syndrome and toxin megacolon. Elderly, debilitated patients in hospitals and nursing homes are particularly vulnerable. A hypervirulent, epidemic strain has been associated with the changing epidemiology and severity of disease. Here, we review the characteristics of the epidemic NAP1, PCR ribotype 027 C. difficile strain that could explain its hypervirulence and epidemic spread.
Abstract: Recent outbreaks of Clostridium difficile infection have been related to the emergence of the NAP1/027 epidemic strain. This strain demonstrates increased virulence and resistance to the C-8-methoxyfluoroquinolones gatifloxacin and moxifloxacin. These antibiotics have been implicated as major C. difficile infection-inducing agents. We investigated by real-time reverse transcription-PCR the impact of subinhibitory concentrations of ampicillin, clindamycin, ofloxacin, and moxifloxacin on the expression of genes encoding three colonization factors, the protease Cwp84, the high-molecular-weight S-layer protein, and the fibronectin-binding protein Fbp68. We have previously shown in six non-NAP1/027 moxifloxacin-susceptible strains that the presence of ampicillin or clindamycin induced an upregulation of these genes, whereas the presence of fluoroquinolones did not. The objective of this study was to analyze the expression of these genes under the same conditions in four NAP1/027 strains, one moxifloxacin susceptible and three moxifloxacin resistant. Two in vitro-selected moxifloxacin-resistant mutants were also analyzed. Moxifloxacin resistance was associated with the Thr82-->Ile substitution in GyrA in all but one of the moxifloxacin-resistant strains. The expression of cwp84 and slpA was strongly increased after culture with ampicillin or clindamycin in NAP1/027 strains. Interestingly, after culture with fluoroquinolones, the expression of cwp84 and slpA was only increased in four moxifloxacin-resistant strains, including the NAP1/027 strains and one of the in vitro-selected mutants. The overexpression of cwp84 was correlated with increased production of the protease Cwp84. The historical NAP1/027 moxifloxacin-susceptible strain and its mutant appear to be differently regulated by fluoroquinolones. Overall, fluoroquinolones appear to favor the expression of some colonization factor-encoding genes in resistant C. difficile strains. The fluoroquinolone resistance of the NAP1/027 epidemic strains could be considered an ecological advantage. This could also increase their colonization fitness and promote the infection.
Abstract: Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. Antibiotics are presumed to disturb the normal intestinal microbiota, leading to depletion of the barrier effect and colonization by pathogenic bacteria. This first step of infection includes adherence to epithelial cells. We investigated the impact of various environmental conditions in vitro on the expression of genes encoding known, or putative, colonization factors: three adhesins, P47 (one of the two S-layer proteins), Cwp66 and Fbp68, and a protease, Cwp84. The conditions studied included hyperosmolarity, iron depletion and exposure to several antibiotics (ampicillin, clindamycin, ofloxacin, moxifloxacin and kanamycin). The analysis was performed on three toxigenic and three non-toxigenic C. difficile isolates using real-time PCR. To complete this work, the impact of ampicillin and clindamycin on the adherence of C. difficile to Caco-2/TC7 cells was analysed. Overall, for the six strains of C. difficile studied, exposure to subinhibitory concentrations (1/2 MIC) of clindamycin and ampicillin led to the increased expression of genes encoding colonization factors. This was correlated with the increased adherence of C. difficile to cultured cells under the same conditions. The levels of gene regulation observed among the six strains studied were highly variable, cwp84 being the most upregulated. In contrast, the expression of these genes was weakly, or not significantly, modified in the presence of ofloxacin, moxifloxacin or kanamycin. These results suggest that, in addition to the disruption of the normal intestinal microbiota and its barrier effect, the high propensity of antibiotics such as ampicillin and clindamycin to induce C. difficile infection could also be explained by their direct role in enhancing colonization by C. difficile.
Abstract: Clostridium difficile pathogenesis is mainly due to toxins A and B. However, the first step of pathogenesis is the colonization process. We evaluated C. difficile surface proteins as vaccine antigens to diminish intestinal colonization in a human flora-associated mouse model. First, we used the flagellar cap protein FliD of C. difficile, in order to test several immunization routes: intranasal, rectal, and intragastric. The rectal route, which is the most efficient, was used to vaccine groups of mice with different antigen combinations. After immunizations, the mice were challenged with the toxigenic C. difficile and a significant statistical difference between the control group and the immunized groups was observed in the colonization levels of C. difficile.
Abstract: The discriminatory potential of a combination of various typing methods was evaluated on a set of 21 Clostridium difficile isolates obtained from symptomatic patients hospitalized in a geriatric unit and 7 non-toxigenic isolates from the same hospital. Isolates were firstly serotyped and toxinotyped. Of the 28 isolates, 19 belonged to serogroup A. PCR-ribotyping and PCR-RFLP on the fliC and slpA genes were then applied to these 19 isolates. The results suggest that the combination of PCR-ribotyping with PCR-RFLP analysis of slpA could be more discriminatory and suitable for studying C. difficile epidemiology.
Abstract: Clostridium difficile pathogenicity is mediated mainly by its A and B toxins, but the colonization process is thought to be a necessary preliminary step in the course of infection. The aim of this study was to characterize the Cwp84 protease of C. difficile, which is highly immunogenic in patients with C. difficile-associated disease and is potentially involved in the pathogenic process. Cwp84 was purified as a recombinant His-tagged protein, and specific antibodies were generated in rabbits. Treatment of multiple-band-containing eluted fractions with a reducing agent or with trypsin led to accumulation of a unique protein species with an estimated molecular mass of 61 kDa, corresponding most likely to mature autoprocessed Cwp84 (mCwp84). mCwp84 showed concentration-dependent caseinolytic activity, with maximum activity at pH 7.5. The Cwp84 activity was inhibited by various cysteine protease inhibitors, such as the specific inhibitor E64, and the anti-Cwp84-specific antibodies. Using fractionation experiments followed by immunoblot detection, the protease was found to be associated with the S-layer proteins, mostly as a nonmature species. Proteolytic assays were performed with extracellular matrix proteins to assess the putative role of Cwp84 in the pathogenicity of C. difficile. No degrading activity was detected with type IV collagen. In contrast, Cwp84 exhibited degrading activity with fibronectin, laminin, and vitronectin, which was neutralized by the E64 inhibitor and specific antibodies. In vivo, this proteolytic activity could contribute to the degradation of the host tissue integrity and to the dissemination of the infection.
Abstract: Sera from patients with Clostridium difficile-associated disease (CDAD) and sera from a control group were analysed by an ELISA to detect antibodies directed against four surface proteins and toxins A and B of C. difficile. The surface proteins were the flagellar cap protein FliD, the flagellin FliC, the adhesin Cwp66 divided into two domains, Cwp66-Nterminal and Cwp66-Cterminal, and the fibronectin-binding protein Fbp68. For each antigen, antibody levels in the CDAD patient group and in the control group were compared. In the CDAD patient group, the mean of the antibody levels decreased from Cwp66-Cterminal to Fbp68, FliD, toxins B and A, Cwp66-Nterminal and finally FliC, suggesting different immunogenic properties among these adhesins. For Cwp66-Nterminal, FliC, FliD and Fbp68, the antibody level observed in the control group was higher than in the CDAD group with a statistically significant difference whereas the antibody level for toxins A and B was not statistically different. In conclusion, this study suggests that during the clinical course of disease, C. difficile adhesins are able to induce an immune response which could play a role in the defence mechanism of the host.
Abstract: Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors, such as surface proteins. Clostridium difficile surface proteins have been identified as (i) adhesins (the flagellar cap protein FliD; the flagellin FliC; and the cell wall protein Cwp 66 with a two domain-structure [Cw 66 N-terminal and Cwp 66 C-terminal domains]) and (ii) protease (the Cwp 84 protein). To address the roles of these proteins in the pathogenesis of Clostridium difficile and to identify vaccine antigen candidates, we analyzed the variability of the proteins and their immunogenicities in 17 patients with C. difficile-associated disease. PCR-restriction fragment length polymorphism analysis of amplified gene products revealed interstrain homogeneity with fliC and fliD, in contrast to cwp 66 genes. Immunoblot analysis showed that FliC and FliD were detected in the majority of isolates. The N-terminal domain of Cwp 66 and Cwp 84 were present in all strains tested, in contrast to the Cwp 66 C-terminal domain, the expression of which was heterogeneous. The 17 sera from the corresponding patients were analyzed by enzyme-linked immunosorbent assay to detect antibodies directed against these proteins. Many patients developed antibodies to FliC, FliD, Cwp 84, and the Cwp 66 C-terminal domain, but not to the Cwp 66 N-terminal domain. In conclusion, this study confirms the expression of these surface proteins of C. difficile during the course of the disease. In addition, the FliC, FliD, and Cwp 84 proteins appeared to be good potential vaccine candidates.
Abstract: Predominant groups of bacteria from a human fecal flora-associated mouse model challenged with amoxicillin-clavulanic acid were quantified with fluorescence in situ hybridization combined with flow cytometry using specific 16S rRNA targeted oligonucleotide probes. This approach provides a useful tool with high throughput to evaluate fecal microflora under antibiotic treatment.
Abstract: Clostridium difficile is an intestinal pathogen, which produces two main virulence factors, the exotoxins A and B. Other bacterial structures have been implicated in the colonization of the gastrointestinal tract, which is the first step of the pathogenic process. C. difficile expresses adherence factors and also, displays some surface-associated proteolytic activity, which could play a role in the physiopathology of this bacterium. The aim of this work was to study the protein named Cwp84 which displays significant homologies with many cysteine proteases. The coding catalytic domain of this protein has been cloned in the expression system pGEX-6P-1, as an in-frame fusion with the gluthatione S-transferase, and subsequently purified. The purified fraction showed proteolytic activity on gelatine and BAPNA, but not on azocoll, suggesting a highly selective substrate specificity. The results obtained from inhibition experiments confirmed that Cwp84 belongs to the cysteine protease family. Cwp84 could play a role in degrading some specific host proteins or in the maturation of surface-associated bacterial proteins.
Abstract: Septicemia due to Neisseria elongata subsp. glycolytica occurs infrequently. We report a case of septicemia in a patient undergoing antimitotic chemotherapy. Gram-negative coccobacilli were isolated from blood cultures. The identity of the isolate by phenotypic methods was uncertain. In contrast, identity was confirmed by 16S ribosomal DNA sequencing, which appeared to be very useful for correct identification.
Abstract: Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins. Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin. To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases. It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli. Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots. The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream. The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence. No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains. The cwp84 gene is strongly transcribed as a monocistronic message. This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C. difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.
Abstract: A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified. The one-copy gene is highly conserved in C. difficile isolates. Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified. Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria. Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin. Indirect immunofluorescence and ELISA were employed to demonstrate that C. difficile could bind both soluble and immobilized fibronectin. With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C. difficile to fibronectin and Vero cells. Furthermore, Vero cells could fix purified membrane-immobilized Fbp68. Thus Fbp68 appears to be one of the several adhesins identified to date in C. difficile.
Abstract: For an in vitro mutant of Streptococcus pneumoniae selected on moxifloxacin four- to eightfold-increased MICs of new fluoroquinolones, only a twofold-increased MIC of ciprofloxacin, and a twofold-decreased MIC of novobiocin were observed. This phenotype was conferred by two mutations: Ser81Phe in GyrA and a novel undescribed His103Tyr mutation in ParE, outside the quinolone resistance-determining region, in the putative ATP-binding site of topoisomerase IV.
Abstract: To evaluate the role of known topoisomerase IV and gyrase mutations in the fluoroquinolone (FQ) resistance of Streptococcus pneumoniae, we transformed susceptible strain R6 with PCR-generated fragments encompassing the quinolone resistance-determining regions (QRDRs) of parC or gyrA from different recently characterized FQ-resistant mutants. Considering the MICs of FQs and the GyrA and/or ParC mutations of the individual transformants, we found three levels of resistance. The first level was obtained when a single target, ParC or GyrA, depending on the FQ, was modified. An additional mutation(s) in a second target, GyrA or ParC, led to the second level. The highest increases in resistance levels were seen for Bay y3118 and moxifloxacin with the transformant harboring a double mutation in both ParC and GyrA. When a single modified target was considered, only the ParC mutation(s) led to an increase in the MICs of pefloxacin and trovafloxacin. In contrast, the GyrA or ParC mutation(s) could lead to increases in the MICs of ciprofloxacin, sparfloxacin, grepafloxacin, Bay y3118, and moxifloxacin. These results suggest that the preferential target of trovafloxacin and pefloxacin is ParC, whereas either ParC or GyrA may both be initial targets for the remaining FQs tested. The contribution of the ParC and GyrA mutations to efflux-mediated FQ resistance was also examined. Active efflux was responsible for two- to fourfold increases in the MICs of ciprofloxacin for the transformants, regardless of the initial FQ resistance levels of the recipients.
Abstract: Transfer of fluoroquinolone (FQ) resistance determinants between Streptococcus pneumoniae and viridans streptococci was explored by transformation in vitro. One-step FQ-resistant parC mutants were selected, and resistance could be transferred from DNA from S. oralis, S. mitis, S. sanguis, and S. constellatus to S. pneumoniae, with frequencies of 10(-3) to <10(-7) in correlation with the homologies of their quinolone resistance determining region sequences (95%, 91%, 85%, and 81%, respectively). Reciprocal transfers of mutated parC from DNA from S. pneumoniae to S. mitis and S. oralis were also observed. Simultaneous transfer of mutated parC and gyrA genes from S. mitis to S. pneumoniae yielded high-level-resistant pneumococcal transformants in one step at low frequencies. The parE-parC region of the type strain S. mitis 103335T had >90% homology with that of S. pneumoniae. The efficient interspecific transfer of quinolone resistance determinants in vitro leads us to anticipate their dissemination in the clinical setting.
Abstract: The accumulation of fluoroquinolones (FQs) was studied in a FQ-susceptible laboratory strain of Streptococcus pneumoniae (strain R6). Uptake of FQs was not saturable, was rapidly reversible, and appeared to occur by passive diffusion. In the presence of glucose, which energizes bacteria, the uptake of FQs decreased. Inhibitors of the proton motive force and ATP synthesis increased the uptake of FQs in previously energized bacteria. Similar results were observed with the various FQs tested and may be explained to be a consequence simply of the pH gradient that exists across the cytoplasmic membrane. From a clinical susceptible strain (strain SPn5907) we isolated in vitro on ciprofloxacin an FQ-resistant mutant (strain SPn5929) for which the MICs of hydrophilic molecules were greater than those of hydrophobic molecules, and the mutant was resistant to acriflavine, cetrimide, and ethidium bromide. Strain SPn5929 showed a significantly decreased uptake of ciprofloxacin, and its determinant of resistance to ciprofloxacin was transferred by transformation to susceptible laboratory strain R6 (strain R6tr5929). No mutations in the quinolone resistance-determining regions of the gyrA and parC genes were found. In the presence of arsenate or carbonyl cyanide m-chlorophenylhydrazone, the levels of uptake of ciprofloxacin by the two resistant strains, SPn5929 and R6tr5929, reached the levels of uptake of their susceptible parents. These results suggest an active efflux of ciprofloxacin in strain SPn5929.
Abstract: The mechanism of high-level fluoroquinolone resistance was studied in strains of Streptococcus pneumoniae, either selected in vitro or isolated from clinical samples. By using DNA from these high-level-resistant strains, low-level-resistant transformants (MIC of pefloxacin, > or = 32 micrograms/ml; MIC of ciprofloxacin, 4 micrograms/ml; MIC of sparfloxacin, 0.50 micrograms/ml) were obtained at high frequencies (ca.10(-2)), while high-level-resistant transformants (MIC of pefloxacin, > or = 64 micrograms/ml; MIC of ciprofloxacin, 16 to 64 micrograms/ml; MIC of sparfloxacin, > or = 8 micrograms/ml) were obtained only at low frequencies (ca.10(-4)). This suggested that mutations in at least two unlinked genes were necessary to obtain high-level resistance. Low-level resistance was associated with ParC mutations (change from Ser to Tyr at position 79 [Ser79Tyr], Ser79Phe, or Asp83Gly). ParC mutations were associated, in high-level-resistant strains and transformants, with alterations in the quinolone resistance-determining region of GyrA (Ser84Tyr, Ser84Phe, and/or Glu88Lys). Low-level resistance was shown to be necessary for expression of the gyrA mutations. No mutation in the region corresponding to the quinolone resistance-determining region of GyrB and no alteration of drug accumulation were found.
