1 Groupe de Recherche sur les Antimicrobiens et les Micro-organismes (GRAM EA 2656, IFR 23), Université de Rouen, Faculté de Médecine-Pharmacie, 22 Boulevard Gambetta, F-76183 Rouen Cedex, France
2 Département de Microbiologie-Immunologie, Faculté de Pharmacie Paris XI, Châtenay-Malabry, France
Correspondence
Ludovic Lemée
ludovic.lemee{at}chu-rouen.fr
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ABSTRACT |
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Nucleotide sequences of the internal fragment genes analysed in this work will be deposited in GenBank under accession numbers DQ102375DQ102379 (for cdtA), DQ117049DQ117053 (for cdtB), DQ117054DQ117074 (for cwp66), DQ117075DQ117103 (for cwp84), DQ117104DQ117132 (for fbp68), DQ117133DQ117161 (for fliC), DQ117162DQ117189 (for fliD), DQ117190DQ117218 (for groEL), DQ117219DQ117240 (for slpA), DQ117241DQ117265 (for tcdA), DQ117266DQ117288 (for tcdB) and DQ117289DQ117311 (for tcdD).
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INTRODUCTION |
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In a previous work, we delineated phylogenetic relationships within C. difficile strains by developing a multilocus sequence typing (MLST) approach (Lemée et al., 2004a). We found that isolates recovered from PMC and from AAD did not cluster in distinct lineages, and thus that no hypervirulent lineage could be characterized within the population of toxigenic human isolates (Lemée et al., 2004a
). Since the previous study was based on the polymorphism of housekeeping genes, which reflects the genetic background of the strains, the aim of the present work was to analyse the polymorphism of virulence-associated genes encoding colonization factors, toxins A and B, the sigma factor TcdD, and binary toxin, and to examine the comparative evolution of these virulence-associated and housekeeping genes within a collection of 29 C. difficile isolates selected as representative of the main clusters previously defined by MLST.
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METHODS |
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Multilocus sequence analysis of virulence-associated genes.
Ten virulence-associated genes were analysed by PCR-sequencing of internal fragments; these included seven colonization factor genes and three genes of the pathogenicity locus (PaLoc). The seven genes encoding colonization factors were as follows (Table 1): cwp84 (Cwp84 protease), cwp66 (Cwp66 adhesin), fbp68 (fibronectin-binding protein), fliC (flagellin protein), fliD (flagellar cap protein), groEL (heat-shock protein) and slpA (S-layer protein); these genes have previously been characterized in C. difficile (Calabi et al., 2001
, 2002
; Cerquetti et al., 2000
; Hennequin et al., 2001
, 2003
; Poilane et al., 1998
; Seddon et al., 1990
; Tasteyre et al., 2001
; Waligora et al., 2001
). Only one copy of each of the seven genes was found on the C. difficile 630 genome (http://www.sanger.ac.uk/). The three PaLoc genes analysed were tcdA (encoding toxin A), tcdB (toxin B) and tcdD (encoding a sigma factor that upregulates the expression of toxins A and B) (Mani & Dupuy, 2001
; Moncrief et al., 1997
). In addition, binary toxin genes were screened by PCR amplification, using primers targeting the binary toxin genes cdtA (forward 5'-TGAACCTGGAAAAGGTGATG-3' and reverse 5'-AGGATTATTTACTGGACCATTTG-3') and cdtB (forward 5'-CTTATTGCAAGTAAATACTGAG-3' and reverse 5'-ACCGGATCTCTTGCTTCAGTC-3') (Stubbs et al., 2000
). When isolates were positive for the presence of cdtA and/or cdtB genes, sequencing of the amplified fragment was performed using the same PCR primers. The genomic locations of all the genes studied here and of the seven housekeeping genes analysed in a previous MLST study (Lemée et al., 2004a
) are shown in Fig. 1
.
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Internal fragments of the selected genes were amplified using the primers listed in Table 1. PCRs were performed on a GeneAmp System 2400 thermal cycler (Applied Biosystems) in a final volume of 50 µl containing 0·5 µM of each primer, 200 µM of each deoxynucleoside triphosphate, and 1·25 U of Taq DNA polymerase (Applied Biosystems) in a 1x amplification buffer (10 mM Tris/HCl, pH 8·3, 50 mM KCl, 2·5 mM MgCl2). The PCR mixtures were heated for 3 min at 95 °C, then a touch-down procedure followed, consisting of 30 s at 95 °C, annealing for 30 s at temperatures decreasing from 65 °C to 55 °C during the first 11 cycles (with 1 °C decremental steps in cycles 1 to 11), and ending with an extension step at 72 °C for 30 s. A total of 40 cycles were performed. PCR products were then purified with a Qiaquick Gel Extraction kit (Qiagen) and sequenced (200500 ng DNA) with PCR forward or reverse primers by using an ABI-PRISM Big Dye terminator sequencing kit on an ABI-PRISM 310 Genetic Analyser (Applied Biosystems). Different sequences of a given locus were given allele numbers, and each unique combination of alleles (multilocus allelic profile) was assigned a sequence type (ST). Single point polymorphisms were assessed by resequencing DNA from two separate PCR experiments.
Computer analysis of sequence data.
Clustering of the 29 isolates from the matrix of pairwise similarities between the allelic profiles was performed using the START program (http://www.mlst.net) by the unweighted pair-group method with arithmetic means (UPGMA). Nucleotide sequences were aligned by using BioEdit Sequence Alignment Editor (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Mean numbers of nucleotide differences between alleles and ratios of nonsynonymous to synonymous substitutions (dN/dS) were calculated to test the degree of selection operating on a locus, using the START program. Gene trees were constructed using the neighbour-joining method and bootstrapping algorithms contained in the MEGA software version 2.1 (Kumar et al., 2001).
The index of association (IA) between alleles (Smith et al., 1993) was used to test for linkage disequilibrium between alleles of the ten loci analysed (colonization factors and PaLoc genes). The observed variance in the distribution of allelic mismatches in all pairwise comparisons of the allelic profiles was compared to that expected in a freely recombining population (linkage equilibrium). The significance of the difference in the observed and expected variances was evaluated by computing the maximum variance in the distribution of allelic mismatches obtained using 100 randomizations of the dataset (http://www.mlst.net).
Finally, the present data, obtained from the multilocus study of virulence-associated genes, were compared to the data obtained from previous MLST analysis of C. difficile housekeeping genes (Lemée et al., 2004a).
Nucleotide sequence accession numbers.
Nucleotide sequences of the internal fragment genes analysed in this work will be deposited in the GenBank database under accession numbers DQ102375DQ102379 (for cdtA), DQ117049DQ117053 (for cdtB), DQ117054DQ117074 (for cwp66), DQ117075DQ117103 (for cwp84), DQ117104DQ117132 (for fbp68), DQ117133DQ117161 (for fliC), DQ117162DQ117189 (for fliD), DQ117190DQ117218 (for groEL), DQ117219DQ117240 (for slpA), DQ117241DQ117265 (for tcdA), DQ117266DQ117288 (for tcdB) and DQ117289DQ117311 (for tcdD).
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RESULTS AND DISCUSSION |
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Overall, six of the ten virulence-associated genes (cwp84, fbp68, groEL, tcdA, tcdB and tcdD) exhibit a moderate polymorphism. Concerning the PaLoc (which includes tcdA, tcdB and tcdD), only twenty different toxinotypes have previously been described in C. difficile (Rupnik et al., 2003). Our data confirm this moderate polymorphism, and give new information about the genetic variability of the tcdD regulator gene, which has never been explored. Concerning colonization factors, the cwp84, fbp68 and groEL genes have been reported as highly conserved, whereas fliC, fliD, and especially cwp66 and slpA, revealed a higher polymorphism (Hennequin et al., 2001
, 2003
; Savariau-Lacomme et al., 2003
). Cwp66 and slpA harbour a two-domain structure, with the cell-surface-exposed domain exhibiting high genetic variability (Calabi et al., 2001
; Savariau-Lacomme et al., 2003
). We encountered difficulties in the amplification of precisely this variable domain in several strains; thus, further studies are needed to design extragenic primers, which would allow a better amplification of the variable domains of the cwp66 and slpA genes.
Multilocus sequence data analysis of virulence-associated genes
Multilocus allelic profile analysis generated 22 different STs among the 29 isolates. The majority of these (21/22 STs) were represented by single isolates. This trend reflects both the great diversity of the isolates, selected from various origins, and the high polymorphism of several of the virulence-associated genes studied in this work. The results of clustering of the allelic profiles by UPGMA are shown in Fig. 2.
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Toxigenic AB+ variant isolates clustered together in ST1 (although recovered from Japan, the USA and from different French hospitals), and defined a specific lineage which diverged by more than 0·9 linkage distance from the other isolates. Toxin A variant (AB+) strains have been reported from a number of countries (Alfa et al., 2000; Borriello et al., 1992
; Brazier et al., 1999
; Kato et al., 1998
; Lyerly et al., 1998
). The majority of them are similar to the reference strain of serogroup F (Delmee et al., 1985
) and share the same genetic changes in the toxin A gene (a 1·7 kb deletion in the repetitive 3'-end domain and a nonsense mutation at position 47) (Rupnik et al., 1998
). They also share the same DNA profile by PCR ribotyping (Stubbs et al., 1999
) and PFGE (Pituch et al., 2001
), although a few different genotypes have recently been described (Rupnik et al., 2003
). These results, together with previous MLST data (Lemée et al., 2004a
), support the hypothesis of a low genetic diversity of AB+ variant strains and of the international spread of this phylogenetic lineage. However, the origin and evolutionary history of this lineage within the C. difficile species need further investigation.
Animal isolates (ST3, ST4, ST14, ST16 and ST21) were intermixed with human isolates, and thus did not constitute a distinct subpopulation. It has been speculated that domestic animals could constitute a reservoir of C. difficile isolates and a potential source for human acquisition (O'Neill et al., 1993). Since the present multilocus analysis of virulence-associated genes, MLST analysis of housekeeping genes (Lemée et al., 2004a
) and PCR ribotyping (Arroyo et al., 2005
) have not characterized any host specificity, we can also presume that animal isolates could constitute a source for human community infections.
Composite sequence-based analysis of virulence-associated genes
Since a single nucleotide substitution in a given locus is sufficient to generate a new allele, some of the loci studied generated numerous different alleles. However, there may be a bias in the estimation of the genetic distance between isolates in this approach, which considers alleles differing by only one point mutation or by multiple polymorphic sites in the same manner. Therefore, clustering based on a 3402 bp composite sequence obtained from the different loci analysed should be more appropriate to characterize the relationships between isolates (Fig. 3). Only twenty-three of the 29 isolates were analysed, because of the lack of toxin gene sequence data for non-toxigenic (AB) isolates and for 1599 and FM18 isolates. The identity between the 23 composite sequences was found to be between 93·3 and 100 %. A dendrogram created from the matrix of pairwise sequence divergences of composite sequences is shown in Fig. 3(A)
. Four divergent lineages were characterized: cluster 2 contains all AB+ variants (including one PMC isolate), cluster 4 contains two PMC isolates (FM16 and 630) and one animal (dog) isolate (654778), cluster 3 corresponds to two human AAD isolates (4984 and CD3), and cluster 1 contains all the remaining isolates, with intermixing of human and animal isolates and of PMC and AAD isolates. Only cluster 4 was not retrieved from composite sequence analysis of housekeeping genes (Fig. 3B
). Overall, clustering generated from composite sequence analysis was congruent with clustering of multilocus allelic profiles.
|
When examining monolocus dendrograms of housekeeping genes and virulence-associated genes (some of which are shown in Fig. 4), topologies derived from all the housekeeping genes and from fbp68, groEL, tcdA, tcdB and tcdB loci were found to be congruent, suggesting a probable co-evolution. Conversely, two discrepancies appeared from the analysis of these monolocus dendrograms. First, fliC (Fig. 4
) and fliD loci exhibited global clustering comparable to housekeeping genes, except for three isolates (630, FM16 and 654778), which were here closely related to the AB+ isolates. Second, cwp66 and slpA (Fig. 4
) loci displayed a much greater polymorphism than the other virulence-associated loci and generated a dendrogram with a topology distinct from the other loci: some isolates were closely related in cwp66 and slpA trees and remote from each other in the other trees (1599 and 630, FM5 and 6761), and conversely, some isolates closely related in other trees were remote in cwp66 and slpA trees (1599 and FM18, 1446 and 669984). This suggests that the evolution of these clusters of genes (fliC/fliD and cwp66/slpA) includes recombinational events and/or strong environmental selective pressure (slpA exhibits a high dN/dS ratio), and that the variability of these genes may allow them to overcome the host immune response. In addition, the present data for the slpA gene suggest that serogroups, which correlate with the polymorphism of the variable domain of slpA (Karjalainen et al., 2002
), depend also on recombinational events and selective pressure.
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Finally, the aim of this multilocus analysis was to study the evolution of virulence-associated genes encoding colonization factors, toxins A and B, the sigma factor TcdD, and binary toxin, and to compare it with the evolution of housekeeping genes. The analysis confirms the clonal population structure of C. difficile, and reveals a co-evolution of several of the virulence-associated genes studied (including toxins A and B and binary toxin genes) with housekeeping genes, whereas flagellin, cwp66 and slpA genes may undergo recombination events and/or environmental selective pressure.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arroyo, L. G., Kruth, S. A., Willey, B. M., Staempfli, H. R., Low, D. E. & Weese, J. S. (2005). PCR ribotyping of Clostridium difficile isolates originating from human and animal sources. J Med Microbiol 54, 163166.
Barbut, F., Mario, N., Meyohas, M. C., Binet, D., Frottier, J. & Petit, J. C. (1994). Investigation of a nosocomial outbreak of Clostridium difficile-associated diarrhoea among AIDS patients by random amplified polymorphic DNA (RAPD) assay. J Hosp Infect 26, 181189.[CrossRef][Medline]
Barbut, F., Decre, D., Lalande, V. & 9 other authors (2005). Clinical features of Clostridium difficile-associated diarrhoea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains. J Med Microbiol 54, 181185.
Bartlett, J. G. (1994). Clostridium difficile: history of its role as an enteric pathogen and the current state of knowledge about the organism. Clin Infect Dis 18, S265S272.[Medline]
Bartlett, J. G., Moon, N., Chang, T. W., Taylor, N. & Onderdonk, A. B. (1978). Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis. Gastroenterology 75, 778782.[Medline]
Bidet, P., Lalande, V., Salauze, B., Burghoffer, B., Avesani, V., Delmee, M., Rossier, A., Barbut, F. & Petit, J. C. (2000). Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile. J Clin Microbiol 38, 24842487.
Bongaerts, G. P. & Lyerly, D. M. (1994). Role of toxins A and B in the pathogenesis of Clostridium difficile disease. Microb Pathog 17, 112.[CrossRef][Medline]
Borriello, S. P. (1998). Pathogenesis of Clostridium difficile infection. J Antimicrob Chemother 41, 1319.[Abstract]
Borriello, S. P., Ketley, J. M., Mitchell, T. J., Barclay, F. E., Welch, A. R., Price, A. B. & Stephen, J. (1987). Clostridium difficile a spectrum of virulence and analysis of putative virulence determinants in the hamster model of antibiotic-associated colitis. J Med Microbiol 24, 5364.[Abstract]
Borriello, S. P., Wren, B. W., Hyde, S., Seddon, S. V., Sibbons, P., Krishna, M. M., Tabaqchali, S., Manek, S. & Price, A. B. (1992). Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile. Infect Immun 60, 41924199.[Abstract]
Brazier, J. S., Stubbs, S. L. & Duerden, B. I. (1999). Prevalence of toxin A negative/B positive Clostridium difficile strains. J Hosp Infect 42, 248249.[Medline]
Calabi, E., Ward, S., Wren, B., Paxton, T., Panico, M., Morris, H., Dell, A., Dougan, G. & Fairweather, N. (2001). Molecular characterization of the surface layer proteins from Clostridium difficile. Mol Microbiol 40, 11871199.[CrossRef][Medline]
Calabi, E., Calabi, F., Phillips, A. D. & Fairweather, N. F. (2002). Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues. Infect Immun 70, 57705778.
Cartmill, T. D., Panigrahi, H., Worsley, M. A., McCann, D. C., Nice, C. N. & Keith, E. (1994). Management and control of a large outbreak of diarrhoea due to Clostridium difficile. J Hosp Infect 27, 115.[CrossRef][Medline]
Cerquetti, M., Molinari, A., Sebastianelli, A., Diociaiuti, M., Petruzzelli, R., Capo, C. & Mastrantonio, P. (2000). Characterization of surface layer proteins from different Clostridium difficile clinical isolates. Microb Pathog 28, 363372.[CrossRef][Medline]
Delmee, M., Homel, M. & Wauters, G. (1985). Serogrouping of Clostridium difficile strains by slide agglutination. J Clin Microbiol 21, 323327.[Medline]
Dhalluin, A., Lemée, L., Pestel-Caron, M., Mory, F., Leluan, G., Lemeland, J. F. & Pons, J. L. (2003). Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene. Syst Appl Microbiol 26, 9096.[CrossRef][Medline]
Eveillard, M., Fourel, V., Barc, M. C., Kerneis, S., Coconnier, M. H., Karjalainen, T., Bourlioux, P. & Servin, A. L. (1993). 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. Mol Microbiol 7, 371381.[Medline]
Hennequin, C., Porcheray, F., Waligora-Dupriet, A., Collignon, A., Barc, M., Bourlioux, P. & Karjalainen, T. (2001). GroEL (Hsp60) of Clostridium difficile is involved in cell adherence. Microbiology 147, 8796.[Medline]
Hennequin, C., Janoir, C., Barc, M. C., Collignon, A. & Karjalainen, T. (2003). Identification and characterization of a fibronectin-binding protein from Clostridium difficile. Microbiology 149, 27792787.[CrossRef][Medline]
Johnson, S. & Gerding, D. N. (1998). Clostridium difficile-associated diarrhea. Clin Infect Dis 26, 10271034.[Medline]
Karjalainen, T., Saumier, N., Barc, M. C., Delmee, M. & Collignon, A. (2002). Clostridium difficile genotyping based on slpA variable region in S-layer gene sequence: an alternative to serotyping. J Clin Microbiol 40, 24522458.
Kato, H., Kato, N., Watanabe, K. & 7 other authors (1998). Identification of toxin A-negative, toxin B-positive Clostridium difficile by PCR. J Clin Microbiol 36, 21782182.
Kelly, C. P., Pothoulakis, C. & LaMont, J. T. (1994). Clostridium difficile colitis. N Engl J Med 330, 257262.
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetics analysis software. Bioinformatics 17, 12441245.
Kyne, L., Warny, M., Qamar, A. & Kelly, C. P. (2000). Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N Engl J Med 342, 390397.
Kyne, L., Warny, M., Qamar, A. & Kelly, C. P. (2001). Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet 357, 189193.[CrossRef][Medline]
Larson, H. E., Price, A. B., Honour, P. & Borriello, S. P. (1978). Clostridium difficile and the aetiology of pseudomembranous colitis. Lancet 1, 10631066.[Medline]
Lemée, L., Dhalluin, A., Pestel-Caron, M., Lemeland, J. F. & Pons, J. L. (2004a). Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. J Clin Microbiol 42, 26092617.
Lemée, L., Dhalluin, A., Testelin, S., Mattrat, M. A., Maillard, K., Lemeland, J. F. & Pons, J. L. (2004b). Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (toxin A), and tcdB (toxin B) genes for toxigenic culture of Clostridium difficile. J Clin Microbiol 42, 57105714.
Lyerly, D. M., Neville, L. M., Evans, D. T. & 7 other authors (1998). Multicenter evaluation of the Clostridium difficile TOX A/B TEST. J Clin Microbiol 36, 184190.
Mani, N. & Dupuy, B. (2001). Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci U S A 98, 58445849.
McEllistrem, M. C., Carman, R. J., Gerding, D. N., Genheimer, C. W. & Zheng, L. (2005). A hospital outbreak of Clostridium difficile disease associated with isolates carrying binary toxin genes. Clin Infect Dis 40, 265272.[CrossRef][Medline]
Moncrief, J. S., Barroso, L. A. & Wilkins, T. D. (1997). Positive regulation of Clostridium difficile toxins. Infect Immun 65, 11051108.[Abstract]
O'Neill, G., Adams, J. E., Bowman, R. A. & Riley, T. V. (1993). A molecular characterization of Clostridium difficile isolates from humans, animals and their environments. Epidemiol Infect 111, 257264.[Medline]
Pituch, H., van den Braak, N., van Leeuwen, W., van Belkum, A., Martirosian, G., Obuch-Woszczatynski, P., Luczak, M. & Meisel-Mikolajczyk, F. (2001). Clonal dissemination of a toxin-A-negative/toxin-B-positive Clostridium difficile strain from patients with antibiotic-associated diarrhea in Poland. Clin Microbiol Infect 7, 442446.[CrossRef][Medline]
Poilane, I., Karjalainen, T., Barc, M. C., Bourlioux, P. & Collignon, A. (1998). Protease activity of Clostridium difficile strains. Can J Microbiol 44, 157161.[CrossRef][Medline]
Rupnik, M., Avesani, V., Janc, M., von Eichel-Streiber, C. & Delmee, M. (1998). A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 36, 22402247.
Rupnik, M., Brazier, J. S., Duerden, B. I., Grabnar, M. & Stubbs, S. L. (2001). Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology 147, 439447.[Medline]
Rupnik, M., Kato, N., Grabnar, M. & Kato, H. (2003). New types of toxin A-negative, toxin B-positive strains among Clostridium difficile isolates from Asia. J Clin Microbiol 41, 11181125.
Savariau-Lacomme, M. P., Lebarbier, C., Karjalainen, T., Collignon, A. & Janoir, C. (2003). Transcription and analysis of polymorphism in a cluster of genes encoding surface-associated proteins of Clostridium difficile. J Bacteriol 185, 44614470.
Seddon, S. V., Hemingway, I. & Borriello, S. P. (1990). Hydrolytic enzyme production by Clostridium difficile and its relationship to toxin production and virulence in the hamster model. J Med Microbiol 31, 169174.[Abstract]
Smith, J. M., Smith, N. H., O'Rourke, M. & Spratt, B. G. (1993). How clonal are bacteria? Proc Natl Acad Sci U S A 90, 43844388.
Spigaglia, P. & Mastrantonio, P. (2002). Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 40, 34703475.
Stubbs, S. L., Brazier, J. S., O'Neill, G. L. & Duerden, B. I. (1999). PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J Clin Microbiol 37, 461463.
Stubbs, S., Rupnik, M., Gibert, M., Brazier, J., Duerden, B. & Popoff, M. (2000). Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile. FEMS Microbiol Lett 186, 307312.[CrossRef][Medline]
Tasteyre, A., Karjalainen, T., Avesani, V., Delmee, M., Collignon, A., Bourlioux, P. & Barc, M. C. (2000). Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups. J Clin Microbiol 38, 31793186.
Tasteyre, A., Barc, M. C., Collignon, A., Boureau, H. & Karjalainen, T. (2001). Role of FliC and FliD flagellar proteins of Clostridium difficile in adherence and gut colonization. Infect Immun 69, 79377940.
Toyokawa, M., Ueda, A., Tsukamoto, H., Nishi, I., Horikawa, M., Sunada, A. & Asari, S. (2003). Pseudomembranous colitis caused by toxin A-negative/toxin B-positive variant strain of Clostridium difficile. J Infect Chemother 9, 351354.[CrossRef][Medline]
Waligora, A. J., Hennequin, C., Mullany, P., Bourlioux, P., Collignon, A. & Karjalainen, T. (2001). Characterization of a cell surface protein of Clostridium difficile with adhesive properties. Infect Immun 69, 21442153.
Zhang, W., Jayarao, B. M. & Knabel, S. J. (2004). Multi-virulence-locus sequence typing of Listeria monocytogenes. Appl Environ Microbiol 70, 913920.
Received 26 April 2005;
revised 27 June 2005;
accepted 4 July 2005.
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