The functional dlt operon of Clostridium butyricum controls the D-alanylation of cell wall components and influences cell septation and vancomycin-induced lysis.

Clostridium butyricum is a Gram-positive bacterium involved in the development of necrotizing enterocolitis (NEC) in preterm infants. To colonize the digestive tract, components of the cell wall of C. butyricum must interact with the intestinal mucosa. The D-alanylation of cell wall components such as teichoic acids results in a net positive charge on the cell wall, which is important for many functions of Gram-positive bacteria. Notably, D-alanylation mediates resistance to antimicrobial peptides and antibiotics. Here, we show that the dlt operon of C. butyricum encodes the enzymes responsible for the D-alanylation of cell wall components and influences the surface properties of the cell wall. We show that the D-alanylation of cell wall components controls the septation of C. butyricum, which is an essential mechanism during vegetative growth. Furthermore, we find that D-alanylation is involved in the resistance of C. butyricum to some cationic antimicrobial peptides (CAMPs) and lysozyme. Finally, we show that the D-alanylation of cell wall components influences vancomycin-induced lysis.

Multi-virulence-locus sequence typing of Staphylococcus lugdunensis generates results consistent with a clonal population structure and is reliable for epidemiological typing.

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis.

Genomic and expression analysis of the vanG-like gene cluster of Clostridium difficile.

Primary antibiotic treatment of Clostridium difficile intestinal diseases requires metronidazole or vancomycin therapy. A cluster of genes homologous to enterococcal glycopeptides resistance vanG genes was found in the genome of C. difficile 630, although this strain remains sensitive to vancomycin. This vanG-like gene cluster was found to consist of five ORFs: the regulatory region consisting of vanR and vanS and the effector region consisting of vanG, vanXY and vanT. We found that 57 out of 83 C. difficile strains, representative of the main lineages of the species, harbour this vanG-like cluster. The cluster is expressed as an operon and, when present, is found at the same genomic location in all strains. The vanG, vanXY and vanT homologues in C. difficile 630 are co-transcribed and expressed to a low level throughout the growth phases in the absence of vancomycin. Conversely, the expression of these genes is strongly induced in the presence of subinhibitory concentrations of vancomycin, indicating that the vanG-like operon is functional at the transcriptional level in C. difficile. Hydrophilic interaction liquid chromatography (HILIC-HPLC) and MS analysis of cytoplasmic peptidoglycan precursors of C. difficile 630 grown without vancomycin revealed the exclusive presence of a UDP-MurNAc-pentapeptide with an alanine at the C terminus. UDP-MurNAc-pentapeptide [d-Ala] was also the only peptidoglycan precursor detected in C. difficile grown in the presence of vancomycin, corroborating the lack of vancomycin resistance. Peptidoglycan structures of a vanG-like mutant strain and of a strain lacking the vanG-like cluster did not differ from the C. difficile 630 strain, indicating that the vanG-like cluster also has no impact on cell-wall composition.

Clostridium difficile has an original peptidoglycan structure with a high level of N-acetylglucosamine deacetylation and mainly 3-3 cross-links.

The structure of the vegetative cell wall peptidoglycan of Clostridium difficile was determined by analysis of its constituent muropeptides with a combination of reverse-phase high pressure liquid chromatography separation of muropeptides, amino acid analysis, mass spectrometry and tandem mass spectrometry. The structures assigned to 36 muropeptides evidenced several original features in C. difficile vegetative cell peptidoglycan. First, it is characterized by a strikingly high level of N-acetylglucosamine deacetylation. In addition, the majority of dimers (around 75%) contains A(2)pm(3) → A(2)pm(3) (A(2)pm, 2,6-diaminopimelic acid) cross-links and only a minority of the more classical Ala(4) → A(2)pm(3) cross-links. Moreover, a significant amount of muropeptides contains a modified tetrapeptide stem ending in Gly instead of D-Ala(4). Two L,D-transpeptidases homologues encoding genes present in the genome of C. difficile 630 and named ldt(cd1) and ldt(cd2), were inactivated. The inactivation of either ldt(cd1) or ldt(cd2) significantly decreased the abundance of 3-3 cross-links, leading to a marked decrease of peptidoglycan reticulation and demonstrating that both ldt(cd1)-and ldt(cd2)-encoded proteins have a redundant L,D-transpeptidase activity. The contribution of 3-3 cross-links to peptidoglycan synthesis increased in the presence of ampicillin, indicating that this drug does not inhibit the L,D-transpeptidation pathway in C. difficile.

Multilocus sequence typing analysis of Staphylococcus lugdunensis implies a clonal population structure.

Staphylococcus lugdunensis is recognized as one of the major pathogenic species within the genus Staphylococcus, even though it belongs to the coagulase-negative group. A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 87 S. lugdunensis isolates from various clinical and geographic sources by DNA sequence analysis of seven housekeeping genes (aroE, dat, ddl, gmk, ldh, recA, and yqiL). The number of alleles ranged from four (gmk and ldh) to nine (yqiL). Allelic profiles allowed the definition of 20 different sequence types (STs) and five clonal complexes. The 20 STs lacked correlation with geographic source. Isolates recovered from hematogenic infections (blood or osteoarticular isolates) or from skin and soft tissue infections did not cluster in separate lineages. Penicillin-resistant isolates clustered mainly in one clonal complex, unlike glycopeptide-tolerant isolates, which did not constitute a distinct subpopulation within S. lugdunensis. Phylogenies from the sequences of the seven individual housekeeping genes were congruent, indicating a predominantly mutational evolution of these genes. Quantitative analysis of the linkages between alleles from the seven loci revealed a significant linkage disequilibrium, thus confirming a clonal population structure for S. lugdunensis. This first MLST scheme for S. lugdunensis provides a new tool for investigating the macroepidemiology and phylogeny of this unusually virulent coagulase-negative Staphylococcus.

Proficiency of PCR in hospital settings for nonculture diagnosis of invasive meningococcal infections.

BACKGROUND: Meningococcal meningitis requires rapid diagnosis and immediate management which is enhanced by the use of PCR for the ascertainment of these infections. However, its use is still restricted to reference laboratories. METHODS: We conducted an inter-laboratory study to assess the implementation and the performance of PCR in ten French hospital settings in 2010. RESULTS: Our data are in favour of this implementation. Although good performance was obtained in identifying Neisseria meningitidis positive samples, the main issue was reported in identifying other species (Streptococcus pneumoniae and Haemophilus influenzae) which are also involved in bacterial meningitis cases. CONCLUSIONS: Several recommendations are required and, mainly, PCR should target the major etiological agents (N. meningitidis, S. pneumonia, and H. influenzae) of acute bacterial meningitis. Moreover, PCR should predict the most frequent serogroups of Neisseria meningitidis according to local epidemiology.

Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens with N-acetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis.

This work reports the characterization of the first known peptidoglycan hydrolase (Acp) produced mainly during vegetative growth of Clostridium perfringens. Acp has a modular structure with three domains: a signal peptide domain, an N-terminal domain with repeated sequences, and a C-terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed lytic activity on the cell walls of several Gram-positive bacterial species. Its hydrolytic specificity was established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling reverse phase-high-pressure liquid chromatography (RP-HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis, which displayed an N-acetylglucosaminidase activity. The study of acp expression showed a constant expression during growth, which suggested an important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the insertion of mobile group II intron strategy (ClosTron). The microscopic examination indicated a lack of vegetative cell separation in the acp mutant strain, as well as the wild-type strain incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The comparative responses of wild-type and acp mutant strains to stresses induced by Triton X-100, bile salts, and vancomycin revealed an implication of Acp in autolysis induced by these stresses. Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative growth and stress-induced autolysis.

Characterization of AtlL, a bifunctional autolysin of Staphylococcus lugdunensis with N-acetylglucosaminidase and N-acetylmuramoyl-l-alanine amidase activities.

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.

Cwp19 Is a Novel Lytic Transglycosylase Involved in Stationary-Phase Autolysis Resulting in Toxin Release in Clostridium difficile.

Clostridium difficile is the major etiologic agent of antibiotic-associated intestinal disease. Pathogenesis of C. difficile is mainly attributed to the production and secretion of toxins A and B. Unlike most clostridial toxins, toxins A and B have no signal peptide, and they are therefore secreted by unusual mechanisms involving the holin-like TcdE protein and/or autolysis. In this study, we characterized the cell surface protein Cwp19, a newly identified peptidoglycan-degrading enzyme containing a novel catalytic domain. We purified a recombinant His6-tagged Cwp19 protein and showed that it has lytic transglycosylase activity. Moreover, we observed that Cwp19 is involved in cell autolysis and that a C. difficilecwp19 mutant exhibited delayed autolysis in stationary phase compared to the wild type when bacteria were grown in brain heart infusion (BHI) medium. Wild-type cell autolysis is correlated to strong alterations of cell wall thickness and integrity and to release of cytoplasmic material. Furthermore, we demonstrated that toxins were released into the extracellular medium as a result of Cwp19-induced autolysis when cells were grown in BHI medium. In contrast, Cwp19 did not induce autolysis or toxin release when cells were grown in tryptone-yeast extract (TY) medium. These data provide evidence for the first time that TcdE and bacteriolysis are coexisting mechanisms for toxin release, with their relative contributions in vitro depending on growth conditions. Thus, Cwp19 is an important surface protein involved in autolysis of vegetative cells of C. difficile that mediates the release of the toxins from the cell cytosol in response to specific environment conditions.IMPORTANCEClostridium difficile-associated disease is mainly known as a health care-associated infection. It represents the most problematic hospital-acquired infection in North America and Europe and exerts significant economic pressure on health care systems. Virulent strains of C. difficile generally produce two toxins that have been identified as the major virulence factors. The mechanism for release of these toxins from bacterial cells is not yet fully understood but is thought to be partly mediated by bacteriolysis. Here we identify a novel peptidoglycan hydrolase in C. difficile, Cwp19, exhibiting lytic transglycosylase activity. We show that Cwp19 contributes to C. difficile cell autolysis in the stationary phase and, consequently, to toxin release, most probably as a response to environmental conditions such as nutritional signals. These data highlight that Cwp19 constitutes a promising target for the development of new preventive and curative strategies.

Comparative in vitro and in vivo study of nine alcohol-based handrubs.

BACKGROUND: Hygienic hand disinfection using alcohol-based handrubs (AHRs) is an alternative method to conventional handwashing in hospital wards. Because a documented choice of such products would consider data from in-care evaluation, we designed a comparative study of 9 AHRs both in vitro and in vivo in actual care conditions. METHODS: The bactericidal activity of AHRs was first measured in vitro against 4 hospital strains exhibiting multiple antibiotic resistance: Acinetobacter baumannii, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter aerogenes. In a second time, AHRs were tested in an intensive care unit for antibacterial activity against the cutaneous flora of personnel hands and for acceptance by the care personnel. RESULTS: The 9 AHRs could be classified in 3 groups on the basis of their in vitro activity: products of the first group showed a bactericidal activity higher than 4 log(10) against the 4 strains. Only 1 of these products presented simultaneously an effective antibacterial activity against the cutaneous flora of care personnel hands and a good acceptance by the care personnel. CONCLUSION: The in vitro study allowed the differentiation of the AHRs tested on the basis of bactericidal activity, but evaluation in an in-care situation allowed further discrimination through both antibacterial activity and acceptance. Thus, the combination of in vitro and in vivo evaluations should provide helpful arguments in the choice of AHRs.

Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene.

Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species. We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species. Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains. A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences. The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific. Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns. A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs). The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences. PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses.

Septic arthritis of the hip caused by Yersinia enterocolitica: a case report.

We report a case of bacteriologically documented hip infection caused by Yersinia enterocolitica. A 67-year-old male with a history of valvular disease was admitted for pain and motion range limitation in the left hip with a fever. No organisms were recovered by needle aspiration, but Yersinia enterocolitica grew in joint fluid obtained by surgical arthrotomy. Investigations of the gastrointestinal tract were normal, and there was no evidence of endocarditis. After 6 weeks of appropriate antibiotic therapy and immobilization with transtibial traction, the clinical and laboratory test abnormalities improved. However, the patient died from an intercurrent condition. Y. enterocolitica, a well-known cause of reactive arthritis, can cause septic arthritis.

Risk factors for invasive pneumococcal disease in HIV-infected adults in France in the highly active antiretroviral therapy era.

Invasive pneumococcal diseases remain frequent and severe in HIV-infected subjects. To identify opportunities for prevention, we assessed risk factors of invasive pneumococcal diseases (IPD) in HIV-infected patients over a 10-year period in France. We performed a retrospective case-control study in a reference centre of HIV management in Paris. All HIV-infected patients having suffered from IPD between 2000 and 2011 were included. Control subjects were HIV-infected with no history of IPD or pneumonia, matched by date of diagnosis of HIV with controls. Two controls were randomly selected for each subject. In all, 42 HIV-infected patients presented 44 IPD episodes during the study period and were compared to 84 controls. In the multivariate analysis, patients with IPD were more likely than controls to have a Charlson Comorbidity Index ≥2 (adjusted OR = 7.07, 95% CI 1.99-25.1, p = 0.003), CD4-cell count <200/cells/µL (aOR = 6.93, 95% CI 1.80-26.7, p = 0.005), HIV-RNA viral load >400 copies/mL (aOR = 5.56, 95% CI 1.58-19.5, p = 0.007) and a non-European origin (aOR = 4.26, 95% CI 1.02-17.9, p = 0.047). HIV-infected patients with a higher burden of comorbidities, uncontrolled HIV replication, low CD4-cell counts and/or of non-European origin are at higher risk of developing IPD. Better screening for and management of HIV infection is necessary to reduce the risk of IPD.

The major autolysin of Staphylococcus lugdunensis, AtlL, is involved in cell separation, stress-induced autolysis and contributes to bacterial pathogenesis.

Staphylococcus lugdunensis is a human skin commensal organism, but it is considered as a virulent Staphylococcus species. In a previous study, we described the first S. lugdunensis autolysin, AtlL. This enzyme displays two enzymatic domains and generates two peptidoglycan hydrolases, an N-acetylmuramoyl-l-alanine amidase and an N-acetylglucosaminidase. In this study, to further investigate the functions of this autolysin, a ΔatlL mutant was constructed. The microscopic examination of the mutant showed cell aggregates and revealed a rough outer cell surface demonstrating, respectively, the roles of AtlL in cell separation and peptidoglycan turnover. This ΔatlL mutant exhibited a lower susceptibility to Triton X-100-induced autolysis assays and appears to be more resistant to cell wall antibiotic-induced lysis and death compared with its parental strain. The atlL mutation affected the biofilm formation capacity of S. lugdunensis. Furthermore, the ΔatlL mutant showed trends toward reduced virulence using the Caenorhabditis elegans model. Overall, AtlL appears as a major cell wall autolysin of S. lugdunensis implicated in cell separation, in stress-induced autolysis and in bacterial pathogenesis.

Multilocus analysis reveals diversity in the genus Tissierella: description of Tissierella carlieri sp. nov. in the new class Tissierellia classis nov.

The genus Tissierella and its relatives Tepidimicrobium, Soehngenia and Sporanaerobacter comprise anaerobic Gram-positive bacilli classified along with Gram-positive cocci in a family with controversial placement designated as incertae sedis XI, in the phylum Firmicutes. We performed a top-down reappraisal of the taxonomy from the phylum to the species level within the genus Tissierella. Reconstruction of high-rank 16S rRNA gene-based phylogenies and their interpretation in a taxonomic purpose allowed defining Tissierellia classis nov. within the phylum Firmicutes while the frames of Tissierellales ord. nov. and Tissierellaceae fam. nov. have to be further strengthened. For species delineation in the genus Tissierella, we studied a population of clinical strains. Beside Tissierella praeacuta, a sub-population of five strains formed a clade in multilocus phylogenies (16S rRNA, cpn60, tpi, recA and spo0A genes). Data such as 16S rRNA gene similarity level, population structure, chromosome organization and murein type indicated that this clade corresponded to a novel species for which the name Tissierella carlieri sp. nov. is proposed, with type strain LBN 295(T)=AIP 268.01(T)=DSM 23816(T)=CCUG 60010(T). Such an approach, associating a phylogenetic reappraisal of high-level taxonomic ranks with weak taxonomic structure and a population study for genus and species delineation is needed to strengthen the taxonomic frame of incertae sedis groups in the phylum Firmicutes.

Characterization of the SigD regulon of C. difficile and its positive control of toxin production through the regulation of tcdR.

Clostridium difficile intestinal disease is mediated largely by the actions of toxins A (TcdA) and B (TcdB), whose production occurs after the initial steps of colonization involving different surface or flagellar proteins. In B. subtilis, the sigma factor SigD controls flagellar synthesis, motility, and vegetative autolysins. A homolog of SigD encoding gene is present in the C.difficile 630 genome. We constructed a sigD mutant in C. difficile 630 ∆erm to analyze the regulon of SigD using a global transcriptomic approach. A total of 103 genes were differentially expressed between the wild-type and the sigD mutant, including genes involved in motility, metabolism and regulation. In addition, the sigD mutant displayed decreased expression of genes involved in flagellar biosynthesis, and also of genes encoding TcdA and TcdB as well as TcdR, the positive regulator of the toxins. Genomic analysis and RACE-PCR experiments allowed us to characterize promoter sequences of direct target genes of SigD including tcdR and to identify the SigD consensus. We then established that SigD positively regulates toxin expression via direct control of tcdR transcription. Interestingly, the overexpression of FlgM, a putative anti-SigD factor, inhibited the positive regulation of motility and toxin synthesis by SigD. Thus, SigD appears to be the first positive regulator of the toxin synthesis in C. difficile.

Prevalence and diversity of Clostridium difficile strains in infants.

During early infancy asymptomatic intestinal colonization by Clostridium difficile is frequent. To update information on infant colonization prevalence and to characterize infant strains, in terms of their virulence factors and their phylogenetic diversity, a prospective screening of C. difficile in the stools of infants 0 to 2 years old was conducted at Jean Verdier Hospital (Hôpital Jean Verdier) over an 18 month period. C. difficile was screened by toxigenic culture, and molecular characterization was performed by PCR-ribotyping and multilocus sequence typing (MLST). The overall C. difficile colonization prevalence was 33.7 % (99/294). The colonization rate by a toxigenic strain was 7.1 % (21/294). Community-acquired C. difficile accounted for 66.7 % (66/99) of cases. Molecular typing was performed on 90 isolates from Jean Verdier Hospital and 8 additional isolates from another hospital in Versailles (Centre Hospitalier de Versailles). Among these isolates, 23 were toxigenic (21 tcdA(+)/tcdB(+) and 2 tcdA(-)/tcdB(+)). All the isolates were negative for the binary toxin genes. Seventeen PCR ribotypes (PRs) were identified, with five PRs accounting for 82.7 % (81/98) of the isolates. MLST generated 15 different sequence types (STs). The predominant genotype, PRJV11-ST38 (33.7 %), included only non-toxigenic strains. Toxigenic strains were distributed in eight genotypes. Neither PR027-ST3, nor PR078/126-ST49 were identified but some PRs/STs corresponded to well-known adult infectious strains. These results indicate that infants are widely colonized by non-toxigenic strains. However, toxigenic adult infectious strains circulate in asymptomatic infants even in the community; thus, infants may be a reservoir for adult infectious strains.

Multilocus sequence typing for Clostridium difficile.

Multilocus sequence typing (MLST), a nucleotide sequence-based characterization of allelic polymorphism of housekeeping genes, has been proposed as a new approach for population and evolutionary genetics and global epidemiology of bacterial pathogens. MLST provides unambiguous sequence data that can be generated from various laboratories and should be shared in a common web database. Here are presented most of materials, methods, and programs or software necessary to perform MLST on Clostridium difficile.We also describe an example of an MLST scheme for C. difficile based on sequence analysis of six housekeeping gene loci and use a set of 74 C. difficile isolates from various hosts, geographic sources, and PCR-toxigenic types (A+B+, A-B+, and A-B-). Thirty-two "sequence types" (ST) are defined from the combination of allelic data, which correlate well with toxigenic types. The estimation of linkage disequilibrium between loci reveals a clonal population structure. Mutational evolution of C. difficile is characterized, with point mutation generating new alleles at a frequency eightfold higher than recombinational exchange. Phylogenetic analysis shows that human and animal isolates do not cluster in distinct lineages, and that no hypervirulent lineage can be characterized within the population of toxigenic human isolates studied (strains from pseudomembranous colitis and antibiotic-associated diarrhea do not cluster in distinct lineages). However, all A-B+ variant isolates belong to a divergent but very homogeneous lineage in the population studied.An MLST database specific for this species is now hosted at the web site of the Institut Pasteur Paris. Since MLST data reflect evolutionary genetics of the species, they could be used as typing markers, possibly in combination with virulence genes data, for long-term global epidemiology of C. difficile.

Tolerance to the glycopeptides vancomycin and teicoplanin in coagulase-negative staphylococci.

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.

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

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.