TcdC Genotypes Associated with Severe TcdC Truncation in an Epidemic Clone and Other Strains of Clostridium difficile
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SUBMITTER: Curry S
PROVIDER: S-EPMC1933044 | biostudies-literature | 2007 Jun
REPOSITORIES: biostudies-literature
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Project description:Severe Clostridium difficile associated disease is associated with outbreaks of the recently described BI/NAP1 epidemic clone. This clone is characterized by an 18-bp deletion in the tcdC gene and increased production of toxins A and B in vitro. TcdC is a putative negative regulator of toxin A&B production. We characterized tcdC genotypes from a collection of C. difficile isolates from a hospital that experienced an outbreak caused by the BI/NAP1 epidemic clone. Sequence analysis of tcdC was performed on DNA samples isolated from 199 toxigenic C. difficile isolates (31% BI/NAP1) from 2001 and 2005. Sequences obtained from 36 (18.6%) isolates predicted wild-type TcdC (232 amino acid residues), whereas 12 (6.1%) isolates had tcdC genotypes with previously described 18- or 39-bp deletions. The remaining isolates comprised 15 unique genotypes. Of these, 5 genotypes contain 18- or 36-bp deletions. Of these five genotypes, one is characterized by a single nucleotide deletion at position 117 resulting in a frameshift that introduces a stop codon at position 196, truncating the predicted TcdC to 65 amino acid residues. All 62 of the isolates in this collection comprising the epidemic clone are characterized by this genotype. This result suggests that severe truncation of TcdC is responsible for the increased toxin production observed in strains belonging to the BI/NAP1 clone and that the 18-bp deletion is probably irrelevant to TcdC function. Further investigations are required to determine the effect of this and other tcdC genotypes on toxin production and clinical disease.
| S-EPMC1828959 | biostudies-literature
Project description:Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.
| S-EPMC3192846 | biostudies-literature
Project description:Clostridium difficile infections are increasing worldwide due to emergence of virulent strains. Infections can result in diarrhea and potentially fatal pseudomembranous colitis. The main virulence factors of C. difficile are clostridial toxins TcdA and TcdB. Transcription of the toxins is positively regulated by the sigma factor TcdR. Negative regulation is believed to occur through TcdC, a proposed anti-sigma factor. Here, we describe the biochemical properties of TcdC to understand the mechanism of TcdC action. Bioinformatic analysis of the TcdC protein sequence predicted the presence of a hydrophobic stretch [amino acids (aa) 30-50], a potential dimerization domain (aa 90-130) and a C-terminal oligonucleotide-binding fold. Gel filtration chromatography of two truncated recombinant TcdC proteins (TcdCΔ1-89 and TcdCΔ1-130) showed that the domain between aa 90 and 130 is involved in dimerization. Binding of recombinant TcdC to single-stranded DNA was studied using a single-stranded Systematic Evolution of Ligands by Exponential enrichment approach. This involved specific binding of single-stranded DNA sequences from a pool of random oligonucleotides, as monitored by electrophoretic-mobility shift assay. Analysis of the oligonucleotides bound showed that the oligonucleotide-binding fold domain of TcdC can bind specifically to DNA folded into G-quadruplex structures containing repetitive guanine nucleotides forming a four-stranded structure. In summary, we provide evidence for DNA binding of TcdC, which suggests an alternative function for this proposed anti-sigma factor.
| S-EPMC3575817 | biostudies-literature
Project description:Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ? 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.
| S-EPMC3122728 | biostudies-literature
Project description:Clostridium difficile infection is the most common cause of severe cases of antibiotic-associated diarrhea (AAD) and is a significant health burden. Recent increases in the rate of C. difficile infection have paralleled the emergence of a specific phylogenetic clade of C. difficile strains (ribotype 027; North American pulsed-field electrophoresis 1 [NAP1]; restriction endonuclease analysis [REA] group BI). Initial reports indicated that ribotype 027 strains were associated with increased morbidity and mortality and might be hypervirulent. Although subsequent work has raised some doubt as to whether ribotype 027 strains are hypervirulent, the strains are considered epidemic isolates that have caused severe outbreaks across the globe. We hypothesized that one factor that could lead to the increased prevalence of ribotype 027 strains would be if these strains had increased competitive fitness compared to strains of other ribotypes. We developed a moderate-throughput in vitro model of C. difficile infection and used it to test competition between four ribotype 027 clinical isolates and clinical isolates of four other ribotypes (001, 002, 014, and 053). We found that ribotype 027 strains outcompeted the strains of other ribotypes. A similar competitive advantage was observed when two ribotype pairs were competed in a mouse model of C. difficile infection. Based upon these results, we conclude that one possible mechanism through which ribotype 027 strains have caused outbreaks worldwide is their increased ability to compete in the presence of a complex microbiota.
| S-EPMC4097623 | biostudies-literature
Project description:BackgroundThe increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production. To assess the role of hyper-production of Toxins A and B in clinical isolates of Clostridium difficile, two NAP1-related and five NAP1 non-related strains were compared.MethodsSequencing was performed on tcdC, tcdR, and tcdE to determine if there were differences that might account for hyper-production of Toxin A and Toxin B in NAP1-related strains. Biological activity of Toxin B was evaluated using the HFF cell CPE assay and Toxin A biological activity was assessed using the Caco-2 Trans-membrane resistance assay.ResultsOur results confirm that Toxin A and Toxin B production in NAP1-related strains and ATCC 43255 occurs earlier in the exponential growth phase compared to most NAP1-nonrelated clinical isolates. Despite the hyper-production observed in ATCC 43255 it had no mutations in tcdC, tcdR or tcdE. Analysis of the other clinical isolates indicated that the kinetics and ultimate final concentration of Toxin A and B did not correlate with the presence or lack of alterations in tcdC, tcdR or tcdE.ConclusionOur data do not support a direct role for alterations in the tcdC gene as a predictor of hyperproduction of Toxin A and B in NAP1-related strains.
| S-EPMC2707377 | biostudies-literature
Project description:Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance-conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.
| S-EPMC3605770 | biostudies-literature
Project description:In the present study we conducted a gene-wide analysis from nine couples of C. difficile strains. Each couple is composed of a high potential virulence (HPV) strain associated with its control (CON) strain. The HPV strains were chosen using two clinical criteria: prevalence and severity. The prevalent strains, defined in this study by an overrepresentation in European hospital, were associated with phylogenetically close control strain but rarely found in hospital. In other hand, the severe strains sampled on patient with very serious symptoms were associated with phylogenetically identical strain but that gave mild symptoms in another patient. The aim of this work was to determined if we could explain at least a part of the clinical criteria differences between these nine couples of strains in the light of their genomic and transcriptomic differences.
2019-09-09 | GSE86612 | GEO
Project description:Clostridium difficile was isolated from 147 of 201 (73%) rectal swabs of piglets from 15 farms of Lower Saxony and North Rhine-Westphalia. In 14 farms, 14 to 100% (mean, 78%) of the animals tested were culture positive. The rate of isolation was 68% postpartum, increased to 94% in animals 2 to 14 days of age, and declined to 0% for animals 49 days of age and older. There was no link between isolation and antibiotic treatment or diarrhea of piglets. Strains were assigned to 10 PCR ribotypes, and up to 4 PCR ribotypes were found to be present at the same time on a farm. The closely related PCR ribotypes 078 (55%) and 126 (20%) were most frequently recovered and were present in 13 of the 14 positive farms. The comparison of multilocus VNTR (variable number of tandem repeats) analysis (MLVA) data from this study and previously published data on human, porcine, and bovine PCR ribotype 078 isolates from 5 European countries revealed genetic differences between strains of different geographic origin and confirmed the relatedness of human and porcine C. difficile isolates. This study demonstrated that the human-pathogenic PCR ribotypes 078 and 126 are predominant in piglets in Germany. The results suggest that presence of C. difficile is correlated with animal age but not with antibiotic treatment or clinical disease. MLVA indicated that strains of the same geographical origin are often genetically related and corroborated the hypothesis of a close epidemiological connection between human and porcine C. difficile isolates.
| S-EPMC3889769 | biostudies-literature
Project description:Clostridium difficile disease has recently increased to become a dominant nosocomial pathogen in North America and Europe, although little is known about what has driven this emergence. Here we show that two epidemic ribotypes (RT027 and RT078) have acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose. RT027 strains contain a single point mutation in the trehalose repressor that increases the sensitivity of this ribotype to trehalose by more than 500-fold. Furthermore, dietary trehalose increases the virulence of a RT027 strain in a mouse model of infection. RT078 strains acquired a cluster of four genes involved in trehalose metabolism, including a PTS permease that is both necessary and sufficient for growth on low concentrations of trehalose. We propose that the implementation of trehalose as a food additive into the human diet, shortly before the emergence of these two epidemic lineages, helped select for their emergence and contributed to hypervirulence.
| S-EPMC5984069 | biostudies-literature