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. INTRODUCTIONSince its recognition as the main cause of pseudomembranous 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 (A2B+ 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.Abbreviations: AAD, antibiotic-associated diarrhoea; MLST, multilocus sequence typing; PaLoc, pathogenicity locus; P...
Tolerance to vancomycin and teicoplanin in 90 clinical isolates of coagulase-negative staphylococci (CoNS) was investigated by time-kill curve methodology. Only six strains, belonging to the Staphylococcus lugdunensis species, exhibited tolerance. The seven other S. lugdunensis strains tested displayed weak susceptibility to the bactericidal activity of glycopeptides compared to the other CoNS. These phenomena are of concern, since S. lugdunensis is recognized as one of the most pathogenic CoNS.Coagulase-negative staphylococci (CoNS) are involved in infections that require bactericidal treatment, such as indwelling foreign body-related infections, endocarditis, and meningitis (4, 10). As CoNS become more resistant to beta-lactams (2), glycopeptides are often considered to be antibiotics of last resort (12). Some investigators, however, have reported glycopeptide tolerance for sporadic CoNS (16,23). Antibiotic tolerance describes a particular "type of resistance" in bacteria capable of surviving, but not growing, in the presence of a normally lethal dose of a given bactericidal antibiotic (20, 21). As early screenings for glycopeptide tolerance in CoNS have been performed by the controversial minimal bactericidal concentration (MBC)/MIC determinations (1,14,19,21), the present study was designed to examine vancomycin and teicoplanin tolerance in a collection of clinically significant CoNS by using the killing curve method, which is considered to be the most reliable method according to the Clinical and Laboratory Standards Institute (CLSI) (formerly NCCLS) (14).An initial set of 79 clinically significant isolates of CoNS from 79 individual patients attending the Rouen University Hospital between January 1999 and April 2001 was studied. Strains were identified to the species level with the ID32Staph system (bioMérieux, Marcy l'Etoile, France) and by a gap gene PCR-restriction fragment length polymorphism assay (24). This set reflected the current epidemiology of CoNS (11), with Staphylococcus epidermidis as a very dominant species (n ϭ 66; 84% of the isolates) and with some less frequently encountered species, i.e., S. hominis (n ϭ 4), S. capitis (n ϭ 3), S. lugdunensis
A gene encoding a putative peptidoglycan hydrolase was identified by sequence similarity searching in the Clostridium difficile 630 genome sequence, and the corresponding protein, named Acd (autolysin of C. difficile) was expressed in Escherichia coli. The deduced amino acid sequence of Acd shows a modular structure with two main domains: an N-terminal domain exhibiting repeated sequences and a C-terminal catalytic domain. The C-terminal domain exhibits sequence similarity with the glucosaminidase domains of Staphylococcus aureus Atl and Bacillus subtilis LytD autolysins. Purified recombinant Acd produced in E. coli was confirmed to be a cell-wall hydrolase with lytic activity on the peptidoglycan of several Gram-positive bacteria, including C. difficile. The hydrolytic specificity of Acd was studied by RP-HPLC analysis and MALDI-TOF MS using B. subtilis cell-wall extracts. Muropeptides generated by Acd hydrolysis demonstrated that Acd hydrolyses peptidoglycan bonds between N-acetylglucosamine and N-acetylmuramic acid, confirming that Acd is an N-acetylglucosaminidase. The transcription of the acd gene increased during vegetative cellular growth of C. difficile 630. The sequence of the acd gene appears highly conserved in C. difficile strains. Regarding deduced amino acid sequences, the C-terminal domain with enzymic function appears to be the most conserved of the two main domains. Acd is the first known autolysin involved in peptidoglycan hydrolysis of C. difficile.
The nucleotide sequence of atlL, a gene encoding a putative Staphylococcus lugdunensis peptidoglycan hydrolase, was determined using degenerate consensus PCR and genome walking. This 3837-bp gene encodes a protein, AtlL, that appears as a putative bifunctional autolysin with a 29-amino acid putative signal peptide and two enzymatic putative centres (N-acetylmuramoyl-l-alanine amidase and N-acetylglucosaminidase) interconnected with three imperfect repeated sequences displaying glycine-tryptophan motifs. In order to determine whether both lytic domains were functional, and verify their exact enzymatic activities, gene fragments harbouring both putative domains, AM (N-acetylmuramoyl-l-alanine amidase enzymatic centre plus two repeated sequences) and GL (N-acetylglucosaminidase enzymatic centre plus one repeated sequence), were isolated, subcloned, and expressed in Escherichia coli. Purified recombinant AM and GL protein truncations exhibited cell wall lytic activity in zymograms performed with cell walls of Micrococcus lysodeikticus, Bacillus subtilis, and S. lugdunensis. AtlL is expressed during the whole growth, with an overexpression in the early-exponential stage. Liquid chromatography-mass spectrometry analysis of muropeptides generated by digestion of B. subtilis cell walls demonstrated the hydrolytic bond specificities and confirmed both of the acetyl domains' activities as predicted by sequence homology data. AtlL is the first autolysin described in S. lugdunensis, with a bifunctional enzymatic activity involved in peptidoglycan hydrolysis.
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