Importance of Toxin A, Toxin B, and CDT in virulence of an epidemic Clostridium difficile strain

Clostridium difficile infection is the main cause of healthcare-acquired diarrhea in the developed world. In addition to the main virulence factors toxin A and B, epidemic, PCR Ribotype 027 strains, such as R20291, produce a third toxin, CDT. To develop effective medical countermeasures, it is important to understand the importance of each toxin. Accordingly, we created all possible combinations of isogenic toxin mutants of R20291 and assessed their virulence. We demonstrated that either toxin A or toxin B alone can cause fulminant disease in the hamster infection model and present tantalizing data that C. difficile toxin may also contribute to virulence

What's a SNP between friends: the influence of single nucleotide polymorphisms on virulence and phenotypes of Clostridium difficile strain 630 and derivatives

Clostridium difficile is a major cause of antibiotic induced diarrhoea worldwide, responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. The two main C. difficile virulence factors are toxins A and B. Isogenic toxin B mutants of two independently isolated erythromycin-sensitive derivatives (630E and 630Δerm) of strain 630 were previously shown to exhibit substantively different phenotypes. Compared to 630, strain 630E and its progeny grow slower, achieve lower final cell densities, exhibit a reduced capacity for spore-formation, produce lower levels of toxin and are less virulent in the hamster infection model. By the same measures, strain 630Δerm and its derivatives more closely mirror the behaviour of 630. Genome sequencing revealed that 630Δerm had acquired seven unique Single Nucleotide Polymorphisms (SNPs) compared to 630 and 630E, while 630E had nine SNPs and a DNA inversion not found in the other two strains. The relatively large number of mutations meant that the identification of those responsible for the altered properties of 630E was not possible, despite the restoration of three mutations to wildtype by allelic exchange and comparative RNAseq analysis of all three strains. The latter analysis revealed large differences in gene expression between the three strains, explaining in part why no single SNP could restore the phenotypic differences. Our findings suggest that strain 630Δerm should be favoured over 630E as a surrogate for 630 in genetic-based studies. They also underline the importance of effective strain curation and the need to genome re-sequence master seed banks wherever possible

Characterization of the impact of rpoB mutations on the in vitro and in vivo competitive fitness of Clostridium difficile and susceptibility to fidaxomicin

ObjectivesTo establish the role of specific, non-synonymous SNPs in the RNA polymerase β subunit (rpoB) gene in reducing the susceptibility of Clostridium difficile to fidaxomicin and to explore the potential in vivo significance of rpoB mutant strains.MethodsAllelic exchange was used to introduce three different SNPs into the rpoB gene of an erythromycin-resistant derivative (CRG20291) of C. difficile R20291. The genome sequences of the created mutants were determined and each mutant analysed with respect to growth and sporulation rates, toxin A/B production and cytotoxicity against Vero cells, and in competition assays. Their comparative virulence and colonization ability was also assessed in a hamster infection model.ResultsThe MIC of fidaxomicin displayed by three mutants CRG20291-TA, CRG20291-TG and CRG20291-GT was substantially increased (>32, 8 and 2 mg/L, respectively) relative to that of the parent strain (0.25 mg/L). Genome sequencing established that the intended mutagenic substitutions in rpoB were the only changes present. Relative to CRG20291, all mutants had attenuated growth, were outcompeted by the parental strain, had lower sporulation and toxin A/B production capacities, and displayed diminished cytotoxicity. In a hamster model, virulence of all three mutants was significantly reduced compared with the progenitor strain, whereas the degree of caecum colonization was unaltered.ConclusionsOur study demonstrates that particular SNPs in rpoB lead to reduced fidaxomicin susceptibility. These mutations were associated with a fitness cost in vitro and reduced virulence in vivo

Molecular genetic typing characteristics of human and animal isolates of Staphylococcus aureus

THESIS 8718Staphylococcus aureus is a commensal and an opportunistic pathogen of both man and animals. It is capable of causing a broad spectrum of diseases, ranging in severity from superficial skin complaints such as boils and impetigo to potentially life-threatening conditions such as toxic shock syndrome and infective endocarditis in man as Avell as mastitis and other diseases in animals. S. aureus can also cause staphylococcal food poisoning in humans which is characterised by the rapid onset and resolution of the symptoms whieh include nausea, vomiting, abdominal pain and, occasionally, diarrhoea. The disease is caused by staphylococcal enterotoxins - secreted proteins which are also known to be superantigenic. As S. aureus can colonise both food handlers and food animals, the source of contamination in an outbreak situation is not always identifiable

Improving the reproducibility of the NAP1/B1/027 epidemic strain R20291 in the hamster model of infection

Comparative analysis of the Clostridium difficile BI/NAP1/027 strain R20291 and ClosTron-derived ermB mutants in the hamster infection model are compromised by the clindamycin susceptibility of the parent. Mutants can appear more virulent. We have rectified this anomaly by genome engineering. The variant created (CRG20291) represents an ideal control strain for virulence assays of ClosTron mutants

Glycin extracts analysis of 630Δerm and R20291 WT, mutant and complemented strains.

<p>(A) Immunoblot analysis with anti-Cwp84 antibodies of glycin extracts, showing complete absence of Cwp84 in the mutants compared to WT and complemented strains. (B) SDS-PAGE of glycin extracts of 630Δerm and R20291 WT, mutant and complemented strains, showing no processing of SlpA precursor in the 630Δ<i>cwp84</i> mutant, and in contrast, an incomplete processing of SlpA in the R20291Δ<i>cwp84</i> mutant. (C) Identification of the HMW-SLP in the glycin extract of the R20291Δ<i>cwp84</i>, showing that a partial processing of SlpA takes place in this mutant even in absence of the Cwp84 protease. Lanes 1, 630Δ<i>erm</i>; lanes 2, 630Δ<i>erm</i>Δ<i>cwp84</i>; lanes 3, 630Δ<i>erm</i>Δ<i>cwp84</i> complemented; lanes 4, R20291; lanes 5, R20291Δ<i>erm</i>Δ<i>cwp84</i>; lanes 6, R20291Δ<i>erm</i>Δ<i>cwp84</i> complemented; MW, molecular weight standard.</p

PCR screening of double crossover candidate clones for complementation of the <i>cwp84</i> gene in <i>C. difficile</i> 630 Δ<i>erm</i> Δ<i>cwp84</i>.

<p>(A) Schematic diagram of the complementation of <i>cwp84</i>, with a single nucleotide change to base 2280 of <i>cwp84</i> from a T to an A, without changing the corresponding valine amino acid residue and at the same time creating a <i>Sca</i>I site. The purpose of this single nucleotide change was to prove the occurrence of the complementation event. (B) PCR screening of candidate clones of the complemented <i>cwp84</i> gene. Primers cwp84-F4 and cwp84-R4 anneal to the internal sequence of the knockout cassette and the downstream sequence of <i>cwp84</i>, respectively, resulting in a 1, 026 bp PCR product from double-crossover complemented clones and wild-type, while no PCR product is expected from Δ<i>cwp84</i> mutants. MW is a 2-Log DNA Ladder (NEB) molecular weight marker, WT is a wild-type <i>C. difficile</i> DNA control, and 1–3 are the candidate clones. All candidates 1 to 3 show the expected complemented 1, 026 bp band, thereby confirmed as <i>cwp84</i> complemented clones, as seen in the wildtype control. (C) PCR products amplified using primers cwp84-F4 and cwp84-R4 from candidates clones and wildtype were analysed by RE digestion with <i>Sca</i>I. PCR products amplified from cwp84 complemented clones were cut into two fragments (786 and 240 bp), whereas PCR products amplified from the wildtype control did not.</p

PyrE ACE correction vectors for <i>C. difficile</i> 630Δ<i>erm</i> (pMTL-YN1) and R20291 (pMTL-YN2).

<p>Both vectors carry identical components between their FseI and SbfI restriction sites. These are: CatP, the <i>catP</i> gene of <i>Clostridium perfringens</i> conferring thiamphenicol resistance; ColE1, the replication region of the <i>E.coli</i> plasmid ColE1, and; TraJ, transfer function of the RP4 <i>oriT</i> region. Plasmids pMTL-YN1C and pMTL-YN2C have an additional segment of DNA inserted between the left-hand homology arm (LHA) and the right-hand homology arm (RHA) which carries: a transcriptional terminator (T1) of the ferredoxin gene of <i>Clostridium pasteurianum</i>; a copy of the <i>lacZ'</i> gene encoding the alpha fragment of the <i>E.coli</i> β-galactosidase, and; a multiple cloning site (MCS) region derived from plasmid pMTL20 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056051#pone.0056051-Chambers1" target="_blank">[19]</a>. Plasmids pMTL-YN1X and pMTL-YN2X differ from pMTL-YN1C and pMTL-YN2C, respectively, in that they carrying the promoter region (P<i>_fdx</i>) of the <i>Clostridium sporogenes</i> ferredoxin gene.</p

Growth of <i>C. difficile</i> 630Δerm strains with mannitol as the sole carbon source.

<p>(A) Clock-wise from top-left, <i>C. difficile</i> 630Δ<i>erm</i> (1) 630Δ<i>erm</i>Δ<i>mtlD</i> mutant (2), and 630Δ<i>erm</i>Δ<i>mtlD</i>-complemented (3) and 630Δ<i>erm</i>Δ<i>mtlD</i>-overexpressed (4) were streaked onto minimal media agar with mannitol as the sole carbon source and incubated for 48 h to observe growth. In contrast to the wild type, complemented and overexpressed strains, no growth was evident for the 630ΔermΔ <i>mtlD</i> mutant. (B) The growth of Δ<i>mtlD</i> was limited in mannitol broth, while growth of the Δ<i>mtlD</i> complemented and <i>mtlD</i> overexpressed strains were restored to wildtype levels. (C) The pH of the media broth showed a dip in pH caused by the fermentation of mannitol for the wildtype, Δ<i>mtlD</i> complemented and Δ<i>mtlD</i> overexpressed strains, which correlate to their growth. The 630 Δ<i>erm</i> Δ<i>mtlD</i> mutant strain grew very weakly in mannitol broth, which was reflected in the sustained pH levels of the media. All experiments were undertaken in triplicate. The data, complete with error bars is provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056051#pone.0056051.s001" target="_blank">Supporting Information File S1</a>.</p

List of oligonucleotides used in this study.

<p>List of oligonucleotides used in this study.</p