Dennis Bakker

Emergence of Clostridium difficile Infection Due to a New Hypervirulent Strain, Polymerase Chain Reaction Ribotype 078

BACKGROUND Since 2005, an increase in the prevalence of Clostridium difficile infection (CDI) due to polymerase chain reaction ribotype 078 has been noticed in The Netherlands. This strain has also been identified as the predominant strain in pigs and calves. METHODS CDI caused by type 078 was studied in relation to CDI caused by the hypervirulent type 027 and by types other than 027 and 078. Human and porcine isolates were further investigated and characterized by multilocus variable number tandem repeat analysis. RESULTS From February 2005 through February 2008, the incidence of type 078 among isolates obtained from 1687 patients increased from 3% to 13%. Compared with patients infected with type 027, patients infected with type 078 were younger (67.4 vs. 73.5 years; P < .01) and more frequently had community-associated disease (17.5% vs. 6.7%; odds ratio, 2.98; 95% confidence interval, 2.11-8.02); rates of severe diarrhea (38.9% vs. 40.0%) and attributable mortality (3.8% vs. 4.0%) were similar in both groups. Compared with patients infected with other types, patients infected with type 078 more frequently received fluoroquinolone therapy (29.4% vs. 19.8%; odds ratio, 2.17; 95% confidence interval, 1.06-4.44). Type 078 isolates contained genes for toxin A, toxin B, binary toxin, and a 39-base pair deletion in toxin regulator gene (tcdC), as well as a point mutation at position 184, resulting in a stop codon. Multilocus variable number tandem repeat analysis of 54 human and 11 porcine isolates revealed 4 clonal complexes containing both porcine and human isolates. CONCLUSIONS CDI due to type 078 and CDI due to type 027 present with similar severity, but CDI due to type 078 affects a younger population and is more frequently community associated. C. difficile type 078 isolates from humans and pigs are highly genetically related.

Relatedness of Human and Animal Clostridium difficile PCR Ribotype 078 Isolates Determined on the Basis of Multilocus Variable-Number Tandem-Repeat Analysis and Tetracycline Resistance

ABSTRACT Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

TcdC does not significantly repress toxin expression in Clostridium difficile 630ΔErm.

In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis, sometimes resulting in colectomy or death. The main virulence factors of C. difficile are toxin A and toxin B. Besides the genes encoding these toxins (tcdA and tcdB), the pathogenicity locus (PaLoc) also contains genes encoding a sigma factor (tcdR) and a putative anti-sigma factor (tcdC). The important role of TcdR as a sigma factor for toxin expression is undisputed, whereas the role of TcdC as an anti-sigma factor, inhibiting toxin expression, is currently the subject of debate. To clarify the role of TcdC in toxin expression, we generated an isogenic ClosTron-based mutant of tcdC in Clostridium difficile strain 630Δ Erm (CT::tcdC) and determined the transcription levels of the PaLoc genes and the expression levels of the toxins in the wild type strain and the tcdC mutant strain. We found only minor differences in transcription levels of the PaLoc genes between the wild type and CT::tcdC strains and total toxin levels did not significantly differ either. These results suggest that in C. difficile 630Δerm TcdC is not a major regulator of toxin expression under the conditions tested.

Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

ABSTRACT In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027.

C. difficile 630Δerm Spo0A Regulates Sporulation, but Does Not Contribute to Toxin Production, by Direct High-Affinity Binding to Target DNA

Clostridium difficile is a Gram positive, anaerobic bacterium that can form highly resistant endospores. The bacterium is the causative agent of C. difficile infection (CDI), for which the symptoms can range from a mild diarrhea to potentially fatal pseudomembranous colitis and toxic megacolon. Endospore formation in Firmicutes, including C. difficile, is governed by the key regulator for sporulation, Spo0A. In Bacillus subtilis, this transcription factor is also directly or indirectly involved in various other cellular processes. Here, we report that C. difficile Spo0A shows a high degree of similarity to the well characterized B. subtilis protein and recognizes a similar binding sequence. We find that the laboratory strain C. difficile 630Δerm contains an 18bp-duplication near the DNA-binding domain compared to its ancestral strain 630. In vitro binding assays using purified C-terminal DNA binding domain of the C. difficile Spo0A protein demonstrate direct binding to DNA upstream of spo0A and sigH, early sporulation genes and several other putative targets. In vitro binding assays suggest that the gene encoding the major clostridial toxin TcdB may be a direct target of Spo0A, but supernatant derived from a spo0A negative strain was no less toxic towards Vero cells than that obtained from a wild type strain, in contrast to previous reports. These results identify for the first time direct (putative) targets of the Spo0A protein in C. difficile and make a positive effect of Spo0A on production of the large clostridial toxins unlikely.

A Novel Secreted Metalloprotease (CD2830) from Clostridium difficile Cleaves Specific Proline Sequences in LPXTG Cell Surface Proteins

Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90β, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro–Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.

Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn6164

BackgroundClostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases.ResultsAnalysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5’and 3’ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype.ConclusionsTn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.

Clostridium difficile TcdC protein binds four-stranded G-quadruplex structures

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.

Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831.

The Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro‐Pro endopeptidase (PPEP‐1). PPEP‐1 cleaves C. difficile cell surface proteins (e.g. CD2831). Here, we confirmed that PPEP‐1 has a unique preference for prolines surrounding the scissile bond. Moreover, we show that it exhibits a high preference for an asparagine at the P2 position and hydrophobic residues at the P3 position. Using a PPEP‐1 knockout C. difficile strain, we demonstrate that the removal of the collagen binding protein CD2831 is fully attributable to PPEP‐1 activity. The PPEP‐1 knockout strain demonstrated higher affinity for collagen type I with attenuated virulence in hamsters.

The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

ABSTRACT In the past decade, Clostridium difficile has emerged as an important gut pathogen. Symptoms of C. difficile infection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that the C. difficile 630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its role in vivo and in vitro through the creation of an isogenic ClosTron-based htrA mutant of C. difficile strain 630Δerm (wild type). In contrast to the attenuated phenotype seen with htrA deletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acute C. difficile infection. Microarray data analysis showed a pleiotropic effect of htrA on the transcriptome of C. difficile, including upregulation of the toxin A gene. In addition, the htrA mutant showed reduced spore formation and adherence to colonic cells. Together, our data show that htrA can modulate virulence in C. difficile.