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Cited by 8Clostridium difficile and Clostridium perfringens researchBiochemical and Structural CharacterizationBacterial Genetics and Biotechnology

Clostridium difficile clade 3 (RT023) have a modified cell surface and contain a large transposable island with novel cargo

Open access
Shaw, Helen Alexandra, Khodadoost, Ladan, Preston, Mark D., Corver, Jeroen, Mullany, Peter, Wren, Brendan W.·2019·Scientific Reports
DOI 10.1038/s41598-019-51628-5PMC PMC6814731
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Abstract

The major global pathogen Clostridium difficile (recently renamed Clostridioides difficile) has large genetic diversity including multiple mobile genetic elements. In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island. These strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile, and loss of the phage protection protein CwpV. A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn 1-3) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome. Novel cargo regions of 023_CTnT include genes encoding a sortase, putative sortase substrates, lantibiotic ABC transporters and a putative siderophore biosynthetic cluster. We demonstrate the excision of 023_CTnT and sub-elements 023_CTn 2 and 023_CTn 3 from the genome of RT023 reference strain CD305 and the transfer of 023_CTn 3 to a non-toxigenic C. difficile strain, which may have implications for the use of non-toxigenic C. difficile strains as live attenuated vaccines. Finally, we show that the genes within the island are expressed in a regulated manner in C. difficile RT023 strains conferring a distinct “niche adaptation”.

AI snapshot

Written from the paper’s full text.

TL;DR

Whole-genome analysis of 86 *Clostridium difficile* clade 3 strains, mostly RT023, identifies a modified cell surface and a large mobile genetic island, 023_CTnT. The island excises, transfers at low frequency to a non-toxigenic strain, and its genes are expressed in a growth-phase-regulated manner.

Key findings

  • Clade 3 strains have a truncated, inactive PPEP-1 protease due to a conserved 2 bp deletion, causing permanent cell-wall association of sortase substrate CD2831; CD305_CD3434 also lacks five of seven PPEP-1 cleavage sites.
  • CwpV is lost in all 86 RT023 strains analysed: a single base-pair deletion in its signal peptide causes a frameshift, and the protein has only two Type III repeats.
  • A 136.4 kb insertion, 023_CTnT, containing 103 predicted coding sequences is present in 80/86 strains analysed; it comprises three putative transposable elements, 023_CTn1–3, with novel cargo including sortase, putative sortase substrates, lantibiotic ABC transporters, and a putative siderophore biosynthetic cluster.
  • 023_CTn3 excises and transfers to non-toxigenic *C. difficile* CD37 at around 10⁻⁷ transconjugants per donor or recipient; 14 island genes were expressed mainly during exponential growth, with diminished or absent expression by stationary phase.

Methods

  • Whole-genome sequencing and bioinformatic analysis of 86 clade 3 *C. difficile* strains, including BLAST and antiSMASH for gene function and biosynthetic cluster prediction.
  • Recombinant PPEP-1 expression in *E. coli*, cell fractionation, immunoblotting, PCR/Sanger sequencing for transposon mobility, and RNA extraction/cDNA PCR for expression analysis.
  • Filter-mating conjugation experiments using CD305 ClosTron mutants as donors and CD37 as recipient, with transconjugants verified by whole-genome sequencing.

Limitations

  • The paper states that a circular PCR product spanning the entire 023_CTnT region could not be consistently amplified with clear sequencing data, suggesting either low-frequency excision or stepwise rather than total excision.
  • The paper states that ClosTrons targeting 023_CTn1 and 023_CTn2 were unsuccessful, and an empty target for 023_CTn2 could not be amplified by PCR.
  • The paper states that the regulation and role of the predicted siderophore in *C. difficile* remains to be determined; evident from the text, expression and transfer experiments were limited to laboratory conditions, three representative RT023 strains for RNA, and one non-toxigenic recipient strain for conjugation.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41598-019-51628-5

Cites

42 of 42 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 28 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Whole genome sequencing of 86 strains from clade 3, predominantly RT023, of C. difficile revealed distinctive surface architecture characteristics.

    “In this study, whole genome sequencing of 86 strains from the poorly characterised clade 3, predominantly PCR ribotype (RT)023, of C. difficile revealed distinctive surface architecture characteristics and a large mobile genetic island.”
    Quote in the paperSubstantial claim
  • Findingown data

    Clade 3 strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile.

    “These strains have a unique sortase substrate phenotype compared with well-characterised strains of C. difficile, and loss of the phage protection protein CwpV.”
    Quote in the paperSubstantial claim
  • Findingown data

    Analysis of the CD305 reference genome showed the presence of CwpV with just two Type III repeats.

    “Analysis of the CD305 reference genome showed the presence of CwpV with just two Type III repeats.”
    Quote in the paperSubstantial claim
  • Findingown data

    There is an absence of proteolytically released CD2831 in the supernatant of CD305 compared with 630.

    “A comparison of 630 and CD305 culture supernatants and whole cell lysates (WCLs) showed an absence of proteolytically released CD2831 in the supernatant of CD305 compared with 630 (Fig. 1d).”
    Quote in the paperSubstantial claim
  • Findingown data

    A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn1-3) is present in 80/86 clade 3 strains analysed.

    “A large genetic insertion (023_CTnT) comprised of three smaller elements (023_CTn1-3) is present in 80/86 strains analysed in this study, with genes common among other bacterial strains in the gut microbiome.”
    Quote in the paperSubstantial claim
  • May combine two claims. 31
  • Classified as probably not a substantial claim. 15

  • May combine two claims.

    Findingcitation

    The 2 bp deletion in CD305 PPEP-1 arises just after the HEXXH catalytic motif, and structural prediction models show a loss of the C-terminal loop.

    “This deletion arises just after the characteristic HEXXH catalytic motif19, with structural prediction models showing a loss of the C-terminal loop (Fig. 1b).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Hensbergen, P. J. et al. A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell surface proteins. Molecular & cellular proteomics: MCP 13, 1231–1244, https://doi.org/10.1074/mcp.M113.034728 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    There is a 2 bp deletion in the reference strain CD305 PPEP-1 homologue (CD305_03825) introducing an in frame stop codon, consistent between all strains in clade 3.

    “There is a 2 bp deletion in the reference strain CD305 PPEP-1 homologue (CD305_03825) introducing an in frame stop codon (Fig. 1a) that was consistent between all strains in this clade (Supplementary Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    023_CTn2 circularises, as shown by PCR, but an empty target could not be amplified.

    “023_CTn2 was shown by PCR to circularise, however an empty target could not be amplified (Fig. 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Whole-genome sequencing analysis of clade 3 strains from the authors' collection and from the literature revealed conserved genetic characteristics that alter the surface architecture of clade 3 and may impact its virulence.

    “We conducted WGS analysis on clade 3 strains from our collection and from the literature, which has revealed conserved genetic characteristics that alter the surface architecture of clade 3 and may impact its virulence.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The empty site is occupied by an imperfect palindrome CACAATGTG, matching the sequence at the 5′ terminus of the CD305 putative transposon within CD305_02397 and the 3′ terminus within CD305_02500, the latter of which contains the perfect palindrome CACATGTG.

    “The empty site is occupied by an imperfect palindrome CACAATGTG, matching the sequence at the 5′ terminus of the CD305 putative transposon within CD305_02397 and the 3′ terminus within CD305_02500, the latter of which contains the perfect palindrome CACATGTG (Fig. 3a).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Most genes within 023_CTnT were expressed well during exponential growth, with CD305_02410, CD305_02437 and CD305_02450 expressed weakly.

    “Figure 6 shows that most of the genes were expressed well during exponential growth, with CD305_02410, CD305_02437 and CD305_02450 expressed weakly.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    023_CTn1 gene CD305_02397 has low sequence identity to the serine recombinase of 630_CTn2 and genes CD305_02422-02426 show significant identity to open reading frames 13-17 of 630_CTn3 (Tn5397) containing conjugation machinery.

    “023_CTn1 gene CD305_02397 has low sequence identity to the serine recombinase of 630_CTn2 and genes CD305_02422–02426 show significant identity to open reading frames 13–17 of 630_CTn3 (Tn5397) containing conjugation machinery (Fig. 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    023_CTn3 circularises and the presence of an empty site was shown by PCR.

    “023_CTn3 was clearly shown to circularise and the presence of an empty site shown by PCR (Fig. 5b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most gene expression was diminished or absent by stationary phase, except for the low expression of CD305_02466 and CD305_02484, suggesting evidence of regulation in 023_CTnT.

    “Meanwhile, most gene expression was diminished or absent by stationary phase, except for the low expression of CD305_02466 and CD305_02484, suggesting evidence of regulation in 023_CTnT as the constitutively expressed core gene slpA was expressed well at both growth phases.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There were minor differences in expression levels between the three strains but no marked differences except for the two non-ribosomal peptide synthetases (CD305_02409, CD305_02410), which show very low expression in CD305 compared with the two other strains, and putative sortase substrate CD305_02437.

    “There were minor differences in expression levels between the three strains but no marked differences except for the two non-ribosomal peptide synthetases (CD305_02409, CD305_02410), which show very low expression in CD305 compared with the two other strains, and putative sortase substrate CD305_02437”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    AntiSMASH analysis revealed the biosynthetic cluster in 023_CTn1 is closely related to a Streptococcus equi cluster producing equibactin, a siderophore for iron acquisition, and a similar cluster within Clostridium kluyveri.

    “AntiSMASH analysis revealed the biosynthetic cluster in 023_CTn1 is closely related to a Streptococcus equi cluster producing equibactin, a siderophore for iron acquisition23, and a similar cluster within Clostridium kluyveri24.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [23] Heather, Z. et al. A novel streptococcal integrative conjugative element involved in iron acquisition. Molecular microbiology 70, 1274–1292, https://doi.org/10.1111/j.1365-2958.2008.06481.x (2008). source

      Not checked in the free run.

    • cites [24] Seedorf, H. et al. The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proceedings of the National Academy of Sciences of the United States of America 105, 2128–2133, https://doi.org/10.1073/pnas.0711093105 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    PCR and Sanger sequencing confirmed the truncation to only two repeats for CwpV and the frame shift within the signal peptide, conserved in all 86 RT023 strains analysed.

    “This, along with Sanger sequencing of the product, confirmed the truncation to only two repeats for CwpV as well as the frame shift within the signal peptide, which was conserved in all 86 RT 023 strains analysed (Supplementary Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The cargo genes of 023_CTnT are unique to clade 3 strains and encode putative proteins with no homology to proteins found in other C. difficile strains.

    “The cargo genes are unique to clade 3 strains and encode putative proteins with no homology to proteins found in other C. difficile strains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Insertion of 023_CTn3 occurred within the 630_CTn7 locus, which in CD37 harbours a transposon with homology to 630_CTn2 that is lost upon acquisition of 023_CTn3.

    “Insertion occurred within the 630_CTn7 locus, which in CD37 harbours a transposon with homology to 630_CTn2 that is lost upon acquisition of 023_CTn3.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    023_CTnT is able to excise from the genome, with evidence of 023_CTn2 and 023_CTn3 circularising, an early step in transfer to other genomes.

    “We have shown here that 023_CTnT is able to excise from the genome, with evidence of 023_CTn2 and 023_CTn3 circularising, an early step in transfer to other genomes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Using ClosTron technology, 023_CTn3 was marked and its transfer to a non-toxigenic strain of C. difficile CD37 was demonstrated, proving that at least part of this region is a mobile element.

    “Using ClosTron technology we were able to mark 023_CTn3 and demonstrate its transfer to a non-toxigenic strain of C. difficile CD37, proving that at least part of this region is a mobile element.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The recipient CD37 does not contain the PaLoc; the PaLoc is replaced by a 115 bp non-coding region in this strain.

  • May combine two claims.

    Findingown data

    RT023 strains also contain a novel genetic element, at least part of which is capable of horizontal gene transfer.

    “They also contain a novel genetic element, at least part of which is capable of horizontal gene transfer.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Findingreasoning

    The authors' work characterised one of the elements that provides this access.

    “Our work characterised one of the elements that provides this access.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bioinformatic analysis of other members of the gut microbiota shows that they have high DNA sequence identity to genes in this element, showing that members of this microbiota have access to a vast gene pool.

    “Furthermore, bioinformatic analysis of other members of the gut microbiota shows that they have high DNA sequence identity to genes in this element, showing that members of this microbiota have access to a vast gene pool.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RT023 strains of C. difficile contain distinctive features on the cell surface, including the loss of CwpV and permanence of collagen-binding sortase substrates.

    “This work has shown that RT023 strains of C. difficile contain distinctive features on the cell surface including the loss of CwpV and permanence of collagen binding sortase substrates.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    PPEP-1 is inactive in RT023, resulting in stable anchoring of sortase substrates to the cell wall.

    “PPEP-1 is inactive in RT023 resulting in stable anchoring of sortase substrates to the cell wall.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Incorporation of a glycosylation cassette into the S-layer locus results in deletion of the cwp2 gene and removal of cwp66 promoters.

    “Incorporation of a glycosylation cassette into the S-layer locus has been shown previously, which results in deletion of the cwp2 gene12, and removal of cwp66 promoters27,28.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [12] Dingle, K. E. et al. Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing. The Journal of infectious diseases 207, 675–686, https://doi.org/10.1093/infdis/jis734 (2013). source

      Not checked in the free run.

    • cites [27] Savariau-Lacomme, M. P., Lebarbier, C., Karjalainen, T., Collignon, A. & Janoir, C. Transcription and analysis of polymorphism in a cluster of genes encoding surface-associated proteins of Clostridium difficile. Journal of bacteriology 185, 4461–4470 (2003). source

      Not checked in the free run.

    • cites [28] Saujet, L., Monot, M., Dupuy, B., Soutourina, O. & Martin-Verstraete, I. The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile. Journal of bacteriology 193, 3186–3196, https://doi.org/10.1128/jb.00272-11 (2011). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Sortases are enzymes that covalently anchor specific protein substrates to the peptidoglycan cell wall or polymerise pili, and are often involved in colonisation and virulence.

    “Sortases are enzymes which covalently anchor specific protein substrates to the peptidoglycan cell wall or polymerise pili31,32 and are often involved in colonisation and virulence33,34.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [31] Dramsi, S. & Bierne, H. Spatial Organization of Cell Wall-Anchored Proteins at the Surface of Gram-Positive Bacteria. Current topics in microbiology and immunology 404, 177–201, https://doi.org/10.1007/82_2016_4 (2017). source

      Not checked in the free run.

    • cites [32] Siegel, S. D., Reardon, M. E. & Ton-That, H. Anchoring of LPXTG-Like Proteins to the Gram-Positive Cell Wall Envelope. Current topics in microbiology and immunology 404, 159–175, https://doi.org/10.1007/82_2016_8 (2017). source

      Not checked in the free run.

    • cites [33] Bierne, H. et al. Inactivation of the srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and affects virulence. Molecular microbiology 43, 869–881 (2002). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    C. difficile is a common coloniser of infants without displaying signs of disease, potentially due to the presence of Bifidobacterium longum.

    “It has been frequently reported that C. difficile is a common coloniser of infants without displaying signs of disease, potentially due to the presence of Bifidobacterium longum30.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [30] Yun, B., Song, M., Park, D. J. & Oh, S. Beneficial Effect of Bifidobacterium longum ATCC 15707 on Survival Rate of Clostridium difficile Infection in Mice. Korean journal for food science of animal resources 37, 368–375, https://doi.org/10.5851/kosfa.2017.37.3.368 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    It is possible that the two additional sortase substrates are substrates for the sortase seen within 023_CTn2.

    “It is possible that they are substrates for the sortase seen within 023_CTn2.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The three sortase substrates are predicted to be collagen-binding proteins and therefore likely to be important in colonisation of the intestine.

    “These three sortase substrates are predicted to be collagen-binding proteins and therefore likely to be important in colonisation of the intestine.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Bifidobacteria are commonly associated with early colonisation of breast-fed infants.

    “Bifidobacteria are commonly associated with early colonisation of breast-fed infants29.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Arboleya, S., Stanton, C., Ryan, C. A., Dempsey, E. & Ross, P. R. Bosom Buddies: The Symbiotic Relationship Between Infants and Bifidobacterium longum ssp. longum and ssp. infantis. Genetic and Probiotic Features. Annual review of food science and technology 7, 1–21, https://doi.org/10.1146/annurev-food-041715-033151 (2016). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    There is evidence that at least 023_CTn2 and 023_CTn3 are capable of excising independently, with 023_CTn3 leaving a clear empty target site.

    “Therefore, there is evidence that at least 023_CTn2 and 023_CTn3 are capable of excising independently, with 023_CTn3 leaving a clear empty target site.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The surface changes in clade 3 could potentially alter colonisation, but this may be counterbalanced by permanent association of core collagen-binding sortase substrates CD2831 and CD3246 on the surface, which may prevent PPEP-1-mediated lifestyle switching.

    “This could potentially alter colonisation, however, this may be counterbalanced by the permanent association of core genes collagen binding sortase substrates CD2831 and CD3246 on the surface of clade 3, which may prevent the hypothesised lifestyle switching through the action of PPEP-1 in these strains7,18.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [7] Hensbergen, P. J. et al. Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831. FEBS letters 589, 3952–3958, https://doi.org/10.1016/j.febslet.2015.10.027 (2015). source

      Not checked in the free run.

    • cites [18] Corver, J., Cordo, V., van Leeuwen, H. C., Klychnikov, O. I. & Hensbergen, P. J. Covalent attachment and Pro-Pro endopeptidase (PPEP-1)-mediated release of Clostridium difficile cell surface proteins involved in adhesion. Molecular microbiology, https://doi.org/10.1111/mmi.13736 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Sortase substrates as cargo on conjugative transposons are common in C. difficile.

    “Sortase substrates as cargo on conjugative transposons is common in C. difficile16”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Brouwer, M. S., Roberts, A. P., Mullany, P. & Allan, E. In silico analysis of sequenced strains of Clostridium difficile reveals a related set of conjugative transposons carrying a variety of accessory genes. Mobile genetic elements 2, 8–12, https://doi.org/10.4161/mge.19297 (2012). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Incorporation of a second sortase enzyme and three sortase substrates in the transposon region may enhance colonisation of clade 3 strains.

    “The incorporation of a second sortase enzyme and three sortase substrates in the transposon region may also enhance the colonisation of these strains.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    It is possible that clade 3 strains acquired transposable elements such as these during colonisation of infants, and acquisition of these genes could enhance long-term colonisation leading to recurrent infections.

    “It is possible that clade 3 strains acquired transposable elements such as these during colonisation of infants and acquisition of these genes could enhance long term colonisation leading to recurrent infections.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The C. kluyveri cluster is adjacent to integrase genes associated with conjugative transposons and is therefore likely to be an element with the potential to transfer between different species of bacteria.

    “The C. kluyveri cluster is adjacent to integrase genes associated with conjugative transposons and is therefore likely to be an element with the potential to transfer between different species of bacteria.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Seedorf, H. et al. The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proceedings of the National Academy of Sciences of the United States of America 105, 2128–2133, https://doi.org/10.1073/pnas.0711093105 (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The inability to amplify an empty target for 023_CTn2 could be due to limited replication of excised 023_CTn2, so that it is present in a higher copy number than its regenerated target and therefore detectable by PCR, whereas the regenerated target is present in too low a concentration to be detected.

    “This could be due to limited replication of excised 023_CTn2 so that it is present in a higher copy number than its regenerated target and therefore detectable by PCR, whereas the regenerated target is present in too low a concentration to be detected.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Due to the nature of the accessory genes present, including those encoding collagen-binding proteins, co-infection with other strains of C. difficile in the intestine could lead to wider dispersion of these genes, with the potential for improved colonisation.

    “Due to the nature of the accessory genes present, including those encoding collagen binding proteins, it is likely that co-infection with other strains of C. difficile in the intestine could lead to wider dispersion of these genes, with the potential for improved colonisation.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CD305_02316 negatively controls the related cluster within Streptococcus equi by inhibiting transcription of synthetase genes.

    “The transcriptional regulator CD305_02316 has been shown to negatively control the related cluster within Streptococcus equi by inhibiting transcription of synthetase genes23”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [23] Heather, Z. et al. A novel streptococcal integrative conjugative element involved in iron acquisition. Molecular microbiology 70, 1274–1292, https://doi.org/10.1111/j.1365-2958.2008.06481.x (2008). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    AbrB is a global transcriptional regulator in Bacillus subtilis that represses the expression of numerous genes at exponential growth phase.

    “AbrB, a global transcriptional regulator in Bacillus subtilis that represses the expression of numerous genes at exponential growth phase38.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [38] Schultz, D., Wolynes, P. G., Ben Jacob, E. & Onuchic, J. N. Deciding fate in adverse times: sporulation and competence in Bacillus subtilis. Proceedings of the National Academy of Sciences of the United States of America 106, 21027–21034, https://doi.org/10.1073/pnas.0912185106 (2009). source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    SNPs and indels differ in the stpK gene between CD37 and CD305, allowing spontaneous mutants of the donor to be distinguished from genuine transconjugants.

    “There are SNPs and indels that differ in this gene between CD37 and CD305 allowing spontaneous mutants of the donor to be distinguished from genuine transconjugants.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Purified gDNA from three independent extractions were analysed by PCR using the high fidelity Phusion polymerase.

    “Purified gDNA from three independent extractions were analysed by PCR using the high fidelity Phusion polymerase (NEB).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Samples were tested for DNA contamination and RNA quality by PCR, nanodrop and bioanalyser.

    “Samples were tested for DNA contamination and RNA quality by PCR, nanodrop and bioanalyser.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    16S PCRs were undertaken on RT+ and RT- samples to show uniform production of cDNA and an absence of gDNA, respectively.

    “16S PCRs were undertaken on RT+ and RT- samples to show uniform production of cDNA and an absence of gDNA respectively.”
    Quote in the paperSubstantial claim
41 of 41

  • The paper relies on this without support.

    Non-toxigenic C. difficile strains are used as live attenuated vaccines.

    “which may have implications for the use of non-toxigenic C. difficile strains as live attenuated vaccines.”

  • The paper relies on this without support.

    The listed bacterial species are found within the human gastrointestinal microbiome.

    “all of which are found within the microbiome of the human gastrointestinal tract.”

  • The paper relies on this without support.

    Clade 3 is a prevalent and important type in Europe, causing clinical symptoms similar to hypervirulent RT027 and RT078 strains and significant recurrence of disease presentation.

    “despite being a prevalent and an important type in Europe causing clinical symptoms similar to hypervirulent strains from RT027 and RT078 and significant recurrence of disease presentation.”

  • The paper relies on this without support.

    The core sortase has specificity for the S/PPKTG motif in substrates.

    “This core sortase has been shown to have specificity for the S/PPKTG motif in substrates”

  • The paper relies on this without support.

    The addition of a second sortase is rare in C. difficile, with the only known duplicate sortase in the core genome within strain 630 (Clade 1).

    “The addition of a second sortase is rare in C. difficile, with the only known duplicate sortase in the core genome to be within strain 630 (Clade 1).”

  • The paper relies on this without support.

    The PaLoc is capable of low frequency transfer between bacterial strains.

    “As the PaLoc is capable of low frequency transfer between bacterial strains (ref)”

  • The paper relies on this without support.

    The recipient CD37 does not contain the PaLoc; the PaLoc is replaced by a 115 bp non-coding region in this strain.

    “the recipient CD37 does not (the PaLoc is replaced by a 115 bp non-coding region in this strain)”
7 of 7

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Shaw et al. identified a large genetic island from the genome of clade 3-associated strains of C. difficile that is predicted to code for the biosynthesis of a Yersiniabactin-like siderophore.

    “However, a recent study conducted by Shaw et al. 137 identified a large genetic island from the genome of clade 3‐associated strains of C. difficile, which, among other peptides, is predicted to code for the biosynthesis of a Yersiniabactin‐like siderophore.”

    From The battle for iron in enteric infections (2020) · cites this paper as [137]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • First recombinase locus tag disagrees with Table 1

    Minor

    The text identifies CD305_02395 as one of the three recombinases in the island, but Table 1 lists CD305_02397 alongside the same other two recombinases. CD305_02395 also falls outside the stated CD305_02397-02499 island range.

  • Anti-His antibody dilution differs between figure and methods

    Minor

    Figure 1 reports a 1:2,000 dilution for the anti-His antibody, whereas the immunoblotting methods specify 1:5000 for that antibody.

2 of 2

Cites

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Cited by

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Related

Authors
Shaw, H. A., Khodadoost, L., Preston, M. D., Corver, J., Mullany, P., & Wren, B. W.
Affiliations
  • London School of Hygiene & Tropical Medicine (GB)
  • University College London (GB)
  • Leiden University Medical Center (NL)
Corresponding author
Shaw, Helen Alexandra
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2019-10-25
Volume
9
Issue
1
Pages
15330-15330
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-019-51628-5
PMC
PMC6814731
OpenAlex
W2981506018
Semantic Scholar
4b94e24f87aed866de048e510f1e1275a0bcc2ec
Cited by
8
References
42
Citations by year
2026: 2 · 2025: 2 · 2024: 2 · 2023: 1 · 2020: 1
FWCI
0.19
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • LSHTM Research Online (London School of Hygiene and Tropical Medicine) (acceptedVersion)
  • Data Archiving and Networked Services (DANS) (submittedVersion)
  • Leiden Repository (Leiden University) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • UCL Discovery (University College London) (submittedVersion)
Funders
Wellcome Trust (102979/Z/13/Z)
Topics
  • Health Sciences › Medicine › Infectious Diseases › Clostridium difficile and Clostridium perfringens research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
Clostridium difficile, clade 3, mobile genetic elements, cell surface, sortase, siderophore biosynthesis, live attenuated vaccine, non-toxigenic strains, gene expression regulation, niche adaptation, gut microbiota
MeSH terms
Amino Acid Sequence, Anaerobiosis, Bacterial Proteins, Base Sequence, Cell Membrane, Cell Wall, DNA Transposable Elements, Genes, Bacterial, Humans, Peptide Hydrolases, Clostridioides difficile, Genomic Islands, Microbiota

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-07.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-07.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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