Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile

Ludovic Lemée1, Ingrid Bourgeois1, Elodie Ruffin1, Anne Collignon2, Jean-François Lemeland1 and Jean-Louis Pons1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
A multilocus sequence analysis of ten virulence-associated genes was performed to study the genetic relationships between 29 Clostridium difficile isolates of various origins, hosts and clinical presentations, and selected from the main lineages previously defined by multilocus sequence typing (MLST) of housekeeping genes. Colonization-factor-encoding genes (cwp66, cwp84, fbp68, fliC, fliD, groEL and slpA), toxin A and B genes (tcdA and tcdB), and the toxin A and B positive regulator gene (tcdD) were investigated. Binary toxin genes (cdtA and cdtB) were also detected, and internal fragments were sequenced for positive isolates. Virulence-associated genes exhibited a moderate polymorphism, comparable to the polymorphism of housekeeping genes, whereas cwp66 and slpA genes appeared highly polymorphic. Isolates recovered from human pseudomembranous colitis cases did not define a specific lineage. The presence of binary toxin genes, detected in five of the 29 isolates (17 %), was also not linked to clinical presentation. Conversely, toxigenic A–B+ isolates defined a very homogeneous lineage, which is distantly related to other isolates. By clustering analysis, animal isolates were intermixed with human isolates. Multilocus sequence analysis of virulence-associated genes is consistent with a clonal population structure for C. difficile and with the lack of host specificity. The data suggest a co-evolution of several of the virulence-associated genes studied (including toxins A and B and the binary toxin genes) with housekeeping genes, reflecting the genetic background of C. difficile, whereas flagellin, cwp66 and slpA genes may undergo recombination events and/or environmental selective pressure.


Abbreviations: AAD, antibiotic-associated diarrhoea; MLST, multilocus sequence typing; PaLoc, pathogenicity locus; PMC, pseudo-membranous colitis; ST, sequence type; UPGMA, unweighted pair-group method with arithmetic means

Nucleotide sequences of the internal fragment genes analysed in this work will be deposited in GenBank under accession numbers DQ102375–DQ102379 (for cdtA), DQ117049–DQ117053 (for cdtB), DQ117054–DQ117074 (for cwp66), DQ117075–DQ117103 (for cwp84), DQ117104–DQ117132 (for fbp68), DQ117133–DQ117161 (for fliC), DQ117162–DQ117189 (for fliD), DQ117190–DQ117218 (for groEL), DQ117219–DQ117240 (for slpA), DQ117241–DQ117265 (for tcdA), DQ117266–DQ117288 (for tcdB) and DQ117289–DQ117311 (for tcdD).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Since its recognition as the main cause of pseudo-membranous colitis (PMC) in 1978, Clostridium difficile has emerged as an important enteropathogen (Bartlett et al., 1978; Johnson & Gerding, 1998; Larson et al., 1978). C. difficile is currently responsible for virtually all cases of PMC and for 10–25 % of antibiotic-associated diarrhoea (AAD) (Bartlett, 1994; Kelly et al., 1994). It is also the leading cause of nosocomial diarrhoea, and many hospital outbreaks have been described throughout the world (Barbut et al., 1994; Cartmill et al., 1994; McEllistrem et al., 2005). The two main virulence factors are exotoxins, toxins A (enterotoxin) and B (cytotoxin), both of which damage the human colonic mucosa and are potent tissue-damaging enzymes (Bongaerts & Lyerly, 1994; Borriello, 1998). Until recently, it was presumed that a strain must produce both toxins to be considered fully pathogenic. However, it is still unclear (i) why strains that do not produce toxin A (A–B+ variant strains) are yet responsible for diarrhoea and even PMC (Alfa et al., 2000; Toyokawa et al., 2003), and (ii) why some patients infected by toxinogenic strains will present a mild diarrhoea, whereas others will present a fulminant PMC. Differences in the levels of production of toxins A and B cannot alone account for the wide spectrum of clinical presentations. Additional virulence factors (i.e. colonization factors and binary toxin) and/or host susceptibility could explain differences in clinical presentations. Adherence determinants are probably largely implicated in the colonization of the gastrointestinal tract by C. difficile, and numerous studies have characterized factors involved in the adherence and colonization processes, such as S-layer proteins (Calabi et al., 2001, 2002; Cerquetti et al., 2000), the Cwp66 adhesin (Waligora et al., 2001), the Fbp68 fibronectin-binding protein (Hennequin et al., 2003), flagella (Tasteyre et al., 2001), the GroEL heat-shock protein (Hennequin et al., 2001) and hydrolytic enzymes (Poilane et al., 1998; Seddon et al., 1990). In addition, some strains of C. difficile also produce an actin-specific ADP-ribosyltransferase (binary toxin), which may act as an additional virulence factor and which may function in synergy with the large clostridial toxins A and B (Barbut et al., 2005).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial isolates.
Twenty-nine C. difficile isolates, selected as representative of the main clusters previously defined by MLST (Lemée et al., 2004a), and recovered from various hosts and geographic sources, were studied. Twenty-four isolates were recovered from human stools: ten from patients with AAD, eight from patients with PMC and four from patients with asymptomatic carriage (the data for two human A–B+ isolates were unknown). Five isolates were recovered from animal hosts suspected to have clostridial intestinal infection. Isolates were identified as C. difficile by Gram stain, colony morphology and fluorescence, API 20A (BioMérieux) biochemical profiles, and for some isolates by sequencing the first 500 bp of 16S rDNA and of an internal fragment of the tpi gene (Dhalluin et al., 2003) to confirm their species identification. The lack of a direct epidemiological link between these 29 strains had previously been confirmed by PCR ribotyping (Lemée et al., 2004a), following a procedure described elsewhere (Bidet et al., 2000). Toxigenic A/B types were determined by multiplex PCR targeting the toxin A and B genes (Lemée et al., 2004b). Among the 29 C. difficile isolates, 17 harboured the tcdA gene (encoding toxin A) and the tcdB gene (encoding toxin B), eight harboured a deleted variant of the tcdA gene and the tcdB gene (A–B+ variants), and four lacked the tcdA and the tcdB genes.

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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Table 1. Genetic polymorphism of the ten virulence-associated genes analysed by MLST

Fragment positions are based on the genome of C. difficile strain 630. dN/dS, ratio of nonsynonymous to synonymous substitutions. I, inosine.

 


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Fig. 1. Genomic locations of the loci analysed in the C. difficile 630 genome. The ten loci analysed in this study are shown in bold; the other genes were analysed previously by MLST (Lemée et al., 2004a). The cdtA and cdtB genes are also shown, but they were found in a complete form in only five of the 29 strains studied.

 
To prepare a DNA sample for PCR amplification, a bacterial colony was taken from blood agar culture and resuspended in 1 ml distilled water in a microcentrifuge tube. The sample was then boiled for 20 min prior to being centrifuged for 2 min to settle bacterial debris, and 10 µl of supernatant, containing the genomic DNA, was used for subsequent PCR amplification.

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 (200–500 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 DQ102375–DQ102379 (for cdtA), DQ117049–DQ117053 (for cdtB), DQ117054–DQ117074 (for cwp66), DQ117075–DQ117103 (for cwp84), DQ117104–DQ117132 (for fbp68), DQ117133–DQ117161 (for fliC), DQ117162–DQ117189 (for fliD), DQ117190–DQ117218 (for groEL), DQ117219–DQ117240 (for slpA), DQ117241–DQ117265 (for tcdA), DQ117266–DQ117288 (for tcdB) and DQ117289–DQ117311 (for tcdD).


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Allelic variation in the virulence-associated genes of C. difficile
Data reporting the allelic variations in the ten virulence-associated genes are summarized in Table 1. The number of individual alleles for each gene ranged from five for tcdB to 17 for cwp84 [some amplification attempts remained unsuccessful for cwp66 (eight isolates), fliD (one isolate), slpA (seven isolates) and tcdB (two isolates), despite the use of various primers spanning the whole gene, and were interpretated as null alleles]. The number of polymorphic sites on a given locus varied from 10 (2·9 %) for tcdB to 387 (98·7 %) for slpA, and the mean genetic distance between alleles of a given locus varied from 0·009 for tcdB to 0·658 for slpA. Most polymorphisms resulted in synonymous substitutions, except for cwp66, tcdB and slpA, for which the ratios of nonsynonymous to synonymous substitutions (dN/dS=0·509, 0·857 and 1·929, respectively) indicate a potential role of environmental selective pressure in their evolution.

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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Fig. 2. Dendrogram showing clustering analysis (UPGMA) of the 29 C. difficile isolates after data compilation from the ten virulence-associated genes. *AAD, antibiotic-associated diarrhoea; D, diarrhoea; PMC, pseudomembranous colitis; AC, asymptomatic carriage; U, unknown. {dagger}H, human; A, animal.

 
Among human isolates, PMC isolates belonged to eight different STs, which did not define PMC-specific clusters in the population studied. In addition, no correlation could be established between virulence-associated gene patterns and clinical presentations. Although toxins A and B are recognized as the main virulence factors of C. difficile (Borriello, 1998), additional virulence factors and/or the host response (Kyne et al., 2000, 2001) could explain the various clinical presentations. Determinants involved in differences in intrinsic virulence between toxigenic strains could be linked to polymorphism in toxin A and B genes (Rupnik et al., 1998), to the level of toxin A and B production (Borriello et al., 1987; Spigaglia & Mastrantonio, 2002), to various patterns of genes encoding colonization factors (Eveillard et al., 1993; Hennequin et al., 2001, 2003; Poilane et al., 1998; Tasteyre et al., 2001; Waligora et al., 2001), or to additional production of a binary toxin (Barbut et al., 2005; McEllistrem et al., 2005). However, despite previous analyses of patterns of colonization factors (Savariau-Lacomme et al., 2003; Tasteyre et al., 2000), toxins A and B (Rupnik et al., 1998), regulators of toxins A and B (Spigaglia & Mastrantonio, 2002), and even binary toxin (Barbut et al., 2005), no correlation has been established to date between genotype and clinical presentation. Since MLST of housekeeping genes also does not define hypervirulent lineages (Lemée et al., 2004a), the genetic determinants of virulence are probably linked not only to the genes encoding virulence factors, but also to genes involved in the regulation of their expression.

Toxigenic A–B+ 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 (A–B+) 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 A–B+ 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 (A–B–) 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 A–B+ 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.



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Fig. 3. Dendrograms showing genetic relationships of C. difficile isolates based on the composite sequence of eight virulence-associated genes (cwp84, fbp68, fliC, fliD, groEL, tcdA, tcdB and tcdD) (A) or six housekeeping genes (B) (Lemée et al., 2004a). In (A), 23 of the 29 isolates are shown (isolates 1446, 669984, FM5 and 650299 lack the pathogenicity locus and tcdA, tcdB, tcdD and tcdB sequences are lacking for the 1599 and FM18 isolates). In (B), the 29 C. difficile isolates are shown.

 
Comparative evolution of virulence-associated genes and housekeeping genes
A quantitative analysis of the linkage between alleles from the different virulence-associated loci was performed by calculating the index of association (IA) (Smith et al., 1993). When all the 29 isolates were included in the analysis, significant linkage disequilibrium was detected (IA, 5·17). At the level of STs (one isolate from each ST, to avoid bias due to a possible epidemic population structure), significant linkage disequilibrium was also detected (IA, 2·01). Previous observations have shown that isolates from diverse geographic origins can be included in the same toxinotype (Rupnik et al., 1998), PCR ribotype (Stubbs et al., 1999) or ST (Lemée et al., 2004a), and that PCR ribotyping and toxinotyping display a strong correlation (Rupnik et al., 2001). All these data are consistent with a strong clonal population structure for C. difficile.

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 A–B+ 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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Fig. 4. Dendrograms showing genetic relationships of the C. difficile isolates based on allele sequences of individual housekeeping loci (aroE, shikimate dehydrogenase, dutA, dUTP pyrophosphatase) and virulence-associated loci. Twenty-five isolates are shown for the tcdA locus (four isolates are A–B–). Twenty-two isolates are shown for the slpA locus (lack of amplification from the other isolates).

 
Detection of the binary toxin genes in the 29 C. difficile isolates
Binary toxin genes were detected in five (17 %) of the 29 isolates. Four were A+B+ toxigenic isolates (three AAD and one PMC human isolates) and one was a non-toxigenic isolate (isolate 650299, recovered from a cow). Both cdtA and cdtB genes were detected in positive isolates. The genetic relationships between the five alleles identified by sequencing of internal fragments and several homologous sequences deposited in GenBank were examined (Fig. 5). Alleles corresponding to the present isolates clustered in two groups (1599/FM18 and CD3/4985/650299), which were also well characterized by housekeeping genes and by virulence-associated genes (except cwp66 and slpA). This also suggests a vertical co-evolution of binary toxin genes with housekeeping genes and several virulence-associated genes, including the PaLoc genes.



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Fig. 5. Dendrograms showing genetic relationships between C. difficile binary toxin cdtA and cdtB alleles characterized in the present work (in bold type) and extracted from GenBank.

 
Multilocus analysis of virulence-associated genes versus MLST of housekeeping genes
The present multilocus analysis of virulence-associated genes was found to be more discriminant than a previous MLST analysis of housekeeping genes (Lemée et al., 2004a). MLST is considered to be a molecular tool for epidemiological purposes, whose main advantage is to provide unambiguous sequence data. However, its discriminatory power is limited by the choice of housekeeping genes analysed. Therefore, multilocus analysis of virulence-associated genes has been proposed as an alternative sequence-typing method (Zhang et al., 2004), reflecting here short-term epidemiology, whereas MLST reflects long-term epidemiology. The selection of two or three highly variable virulence-associated genes, such as slpA, cwp66, and/or fliC, would be well suited to short-term epidemiological sequence-based analysis of C. difficile.

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.


   ACKNOWLEDGEMENTS
 
The authors thank Karine Maillard (Caen, France), Frederic Barbut (Paris, France), Francine Mory (Nancy, France) and Haru Kato (Tokyo, Japan) for supplying strains.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 26 April 2005; revised 27 June 2005; accepted 4 July 2005.



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