Multilocus Sequence Typing Analysis of Human and Animal
            <i>Clostridium difficile</i>
            Isolates of Various Toxigenic Types

Lemée, Ludovic; Dhalluin, Anne; Pestel-Caron, Martine; Lemeland, Jean-François; Pons, Jean-Louis

doi:10.1128/jcm.42.6.2609-2617.2004

Cited by 133 publications

(119 citation statements)

References 55 publications

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“…C. difficile 630 (Sebaihia et al, 2006) and C. difficile 630Derm (Hussain et al, 2005), which is a spontaneously cured derivative of strain 630 and allows selection of ClosTron mutants, were used in all experiments. An additional set of 83 C. difficile isolates selected as representative of the main clusters that we previously defined by multilocus sequence typing (MLST) (Lemee et al, 2004;Lemée & Pons, 2010), and recovered from various hosts and geographical sources, were used to study the phylogenetic origin of the vanG-like operon. All C. difficile strains were cultured on blood agar (Oxoid), BHI agar (Difco) or BHI broth (Difco) at 37 uC in an anaerobic environment [80 % N 2 , 10 % CO 2 , and 10 % (v/v) H 2 ].…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2013

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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 vanGlike cluster also has no impact on cell-wall composition.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2013

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“…The advantages of MLST for local and global epidemiology have been extensively discussed elsewhere (Cooper & Feil, 2004;Enright & Spratt, 1999;Maiden et al, 1998;Spratt, 1999). MLST has been successfully used for the determination of clonal complexes (CC) of several human and animal pathogens (Dingle et al, 2001;Enright et al, 2001;Feavers et al, 1999;Heym et al, 2002;Homan et al, 2002;King et al, 2002;Kriz et al, 2002;Lemee et al, 2004;Nallapareddy et al, 2002;Noller et al, 2003;Shi et al, 1998;van Loo et al, 2002;Wang et al, 2003), including Haemophilus influenzae (Meats et al, 2003). Since the genomic sequence of H. parasuis is not available, we used primers designed for H. influenzae (Meats et al, 2003), universal primers (Christensen et al, 2004), or primers designed to areas of homology of the selected genes in other bacteria, including Pasteurellaceae.…”

Section: Introductionmentioning

confidence: 99%

Study of the population structure of Haemophilus parasuis by multilocus sequence typing

2006

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Haemophilus parasuis is the aetiological agent of Glä sser's disease in swine. In addition, this bacterium causes other clinical outcomes and can also be isolated from the upper respiratory tract of healthy pigs. Isolates of H. parasuis differ in phenotypic features (e.g. protein profiles, colony morphology or capsule production) and pathogenic capacity. Differences among strains have also been demonstrated at the genetic level. Several typing methods have been used to classify H. parasuis field strains, but they had resolution or implementation problems. To overcome these limitations, a multilocus sequence typing (MLST) system, using partial sequences of the house-keeping genes mdh, 6pgd, atpD, g3pd, frdB, infB and rpoB, was developed. Eleven reference strains and 120 field strains were included in this study. The number of alleles per locus ranged from 14 to 41, 6pgd being the locus with the highest diversity. The high genetic heterogeneity of this bacterium was confirmed with MLST, since the strains were divided into 109 sequence types, and only 13 small clonal complexes were detected by the Burst algorithm. Further analysis by unweighted-pair group method with arithmetic mean (UPGMA) identified six clusters. When the clinical background of the isolates was examined, one cluster was statistically associated with nasal isolation (putative non-virulent), while another cluster showed a significant association with isolation from clinical lesions (putative virulent). The remaining clusters did not show a statistical association with the clinical background of the isolates. Finally, although recombination among H. parasuis strains was detected, two divergent branches were found when a neighbour-joining tree was constructed with the concatenated sequences. Interestingly, one branch included almost all isolates of the putative virulent UPGMA cluster. INTRODUCTIONHaemophilus parasuis is a member of the family Pasteurellaceae and the causative agent of Glässer's disease in pigs, which is pathologically characterized by fibrinous to fibrinopurulent polyserositis and polyarthritis (RappGabrielson et al., 2006). In addition to Glässer's disease, H. parasuis produces other clinical outcomes, such as pneumonia, and colonizes the upper respiratory tract of healthy animals (Rapp-Gabrielson et al., 2006). Although it is commonly accepted that one strain is responsible for each clinical outbreak, diagnosis of H. parasuis infection is complicated by the fact that it is usual to detect several strains in a farm and even within a single animal. Therefore, it is essential to determine the causative strain by its isolation from organs with the characteristic lesions of the disease.Differences among strains in phenotypic and genotypic characteristics have been reported, although no clear association with virulence could be determined (Oliveira & Pijoan, 2004;Rapp-Gabrielson et al., 2006). However, several studies have confirmed that different strains of H. parasuis have different pathogenic capacity (Kielstein & Rapp-Gabrielson, 199...

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“…The Maxima SYBR Green qPCR Master Mix (Fermentas) was used according to the manufacturer's instructions. The number of bacteria was evaluated by performing qPCR of the C. difficile-specific housekeeping gene triose-phosphate isomerase (tpi) (Lemee et al, 2004). Primers designed for tpi were: forward, 59-TATGGACT-ATGTTGTAATAGGAC-39; and reverse, 59-CATAATATTGGGTC-TATTCCTAC-39.…”

Section: Introductionmentioning

confidence: 99%

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

Dingle

Mulvey

Humphries

et al. 2010

Journal of Medical Microbiology

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Herein we describe a real-time quantitative PCR assay for evaluating the adherence of Clostridium difficile to differentiated human intestinal Caco-2 cells. Our investigations demonstrated that the method, employing the C. difficile-specific triose-phosphate isomerase gene, is as reliable but less time-consuming than counting c.f.u. We conclude that the method will be useful for evaluating the role of host cell adherence in the pathogenesis of C. difficile infection. INTRODUCTIONClostridium difficile is a Gram-positive anaerobic microorganism that is the most important cause of antibioticassociated diarrhoea, now generally referred to as C. difficile infection (CDI), in humans. To cause clinical illness, C. difficile first penetrates the mucus layer covering the epithelial surface of the gastrointestinal (GI) tract; a strategy that is most likely facilitated by flagella and proteases (Deneve et al., 2009;Tasteyre et al., 2001). Once established in the GI tract, C. difficile expresses two large exotoxins, toxin A (TcdA) and toxin B (TcdB) (Jank & Aktories, 2008;Voth & Ballard, 2005). Delivery of TcdA and TcdB to the cytoplasmic compartment of epithelial cells then leads to glucosylation of Rho family GTPases, resulting in disorganization of the host cell cytoskeleton (Deneve et al., 2009). This results in the loosening of tight junctions between adjacent intestinal epithelial cells ultimately leading to inflammation and diarrhoea.Although TcdA and TcdB represent primary virulence factors, whether adherence to host epithelial cells is also important in the C. difficile pathogenic strategy is less clear. However, C. difficile adherence to epithelial cells in vitro has been reported to be mediated by several colonization factors, including the low-and high-molecular-mass Slayer proteins which are derived by post-translational cleavage of the S-layer precursor protein, SlpA (Calabi et al., 2001). The high-molecular-mass S-layer protein is largely responsible for C. difficile binding to both human GI tissues and to components of the extracellular matrix (Calabi et al., 2002). In addition, Cwp84, a putative colonization factor and cysteine protease, has recently been implicated in a process whereby SlpA is cleaved into lowand high-molecular-mass counterparts (Janoir et al., 2007;Kirby et al., 2009). Several other cell-surface colonization factors including Cwp66, Fbp68 and GroEL, may also all play a role in C. difficile adherence to intestinal epithelial cells (Hennequin et al., , 2003Waligora et al., 2001). Moreover, the flagellar proteins FliC (flagellin) and FliD (flagellar cap protein) bind to axenic mouse mucus possibly facilitating the initial penetration of the intestinal mucus layer (Tasteyre et al., 2001). CDI patients are known to produce serum antibodies to one or more of these cellsurface proteins during infection, emphasizing their potential role in pathogenesis and generating a protective host immune response (Pechine et al., 2005;Wright et al., 2008).Several in vitro C. difficile adherence assays have be...

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Multilocus Sequence Typing Analysis of Human and Animal Clostridium difficile Isolates of Various Toxigenic Types

Cited by 133 publications

References 55 publications

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

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

Study of the population structure of Haemophilus parasuis by multilocus sequence typing

A real-time quantitative PCR assay for evaluating Clostridium difficile adherence to differentiated intestinal Caco-2 cells

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