Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of <italic>Clostridium difficile</italic></title><author><name sortKey="Carter, Glen P" sort="Carter, Glen P" uniqKey="Carter G" first="Glen P." last="Carter">Glen P. Carter</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Douce, Gillian R" sort="Douce, Gillian R" uniqKey="Douce G" first="Gillian R." last="Douce">Gillian R. Douce</name><affiliation><nlm:aff id="aff2"><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Govind, Revathi" sort="Govind, Revathi" uniqKey="Govind R" first="Revathi" last="Govind">Revathi Govind</name><affiliation><nlm:aff id="aff3"><addr-line>Division of Biology, Kansas State University, Manhattan, Kansas, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Howarth, Pauline M" sort="Howarth, Pauline M" uniqKey="Howarth P" first="Pauline M." last="Howarth">Pauline M. Howarth</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mackin, Kate E" sort="Mackin, Kate E" uniqKey="Mackin K" first="Kate E." last="Mackin">Kate E. Mackin</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Spencer, Janice" sort="Spencer, Janice" uniqKey="Spencer J" first="Janice" last="Spencer">Janice Spencer</name><affiliation><nlm:aff id="aff2"><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Buckley, Anthony M" sort="Buckley, Anthony M" uniqKey="Buckley A" first="Anthony M." last="Buckley">Anthony M. Buckley</name><affiliation><nlm:aff id="aff2"><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Antunes, Ana" sort="Antunes, Ana" uniqKey="Antunes A" first="Ana" last="Antunes">Ana Antunes</name><affiliation><nlm:aff id="aff4"><addr-line>Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kotsanas, Despina" sort="Kotsanas, Despina" uniqKey="Kotsanas D" first="Despina" last="Kotsanas">Despina Kotsanas</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Infectious Diseases, Southern Health, Monash Medical Centre, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jenkin, Grant A" sort="Jenkin, Grant A" uniqKey="Jenkin G" first="Grant A." last="Jenkin">Grant A. Jenkin</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Infectious Diseases, Southern Health, Monash Medical Centre, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Dupuy, Bruno" sort="Dupuy, Bruno" uniqKey="Dupuy B" first="Bruno" last="Dupuy">Bruno Dupuy</name><affiliation><nlm:aff id="aff4"><addr-line>Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Rood, Julian I" sort="Rood, Julian I" uniqKey="Rood J" first="Julian I." last="Rood">Julian I. Rood</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lyras, Dena" sort="Lyras, Dena" uniqKey="Lyras D" first="Dena" last="Lyras">Dena Lyras</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22022270</idno><idno type="pmc">3192846</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192846</idno><idno type="RBID">PMC:3192846</idno><idno type="doi">10.1371/journal.ppat.1002317</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">002C57</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">002C57</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of <italic>Clostridium difficile</italic></title><author><name sortKey="Carter, Glen P" sort="Carter, Glen P" uniqKey="Carter G" first="Glen P." last="Carter">Glen P. Carter</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Douce, Gillian R" sort="Douce, Gillian R" uniqKey="Douce G" first="Gillian R." last="Douce">Gillian R. Douce</name><affiliation><nlm:aff id="aff2"><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Govind, Revathi" sort="Govind, Revathi" uniqKey="Govind R" first="Revathi" last="Govind">Revathi Govind</name><affiliation><nlm:aff id="aff3"><addr-line>Division of Biology, Kansas State University, Manhattan, Kansas, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Howarth, Pauline M" sort="Howarth, Pauline M" uniqKey="Howarth P" first="Pauline M." last="Howarth">Pauline M. Howarth</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Mackin, Kate E" sort="Mackin, Kate E" uniqKey="Mackin K" first="Kate E." last="Mackin">Kate E. Mackin</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Spencer, Janice" sort="Spencer, Janice" uniqKey="Spencer J" first="Janice" last="Spencer">Janice Spencer</name><affiliation><nlm:aff id="aff2"><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Buckley, Anthony M" sort="Buckley, Anthony M" uniqKey="Buckley A" first="Anthony M." last="Buckley">Anthony M. Buckley</name><affiliation><nlm:aff id="aff2"><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></nlm:aff></affiliation></author><author><name sortKey="Antunes, Ana" sort="Antunes, Ana" uniqKey="Antunes A" first="Ana" last="Antunes">Ana Antunes</name><affiliation><nlm:aff id="aff4"><addr-line>Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Kotsanas, Despina" sort="Kotsanas, Despina" uniqKey="Kotsanas D" first="Despina" last="Kotsanas">Despina Kotsanas</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Infectious Diseases, Southern Health, Monash Medical Centre, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Jenkin, Grant A" sort="Jenkin, Grant A" uniqKey="Jenkin G" first="Grant A." last="Jenkin">Grant A. Jenkin</name><affiliation><nlm:aff id="aff5"><addr-line>Department of Infectious Diseases, Southern Health, Monash Medical Centre, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Dupuy, Bruno" sort="Dupuy, Bruno" uniqKey="Dupuy B" first="Bruno" last="Dupuy">Bruno Dupuy</name><affiliation><nlm:aff id="aff4"><addr-line>Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France</addr-line></nlm:aff></affiliation></author><author><name sortKey="Rood, Julian I" sort="Rood, Julian I" uniqKey="Rood J" first="Julian I." last="Rood">Julian I. Rood</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lyras, Dena" sort="Lyras, Dena" uniqKey="Lyras D" first="Dena" last="Lyras">Dena Lyras</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS Pathogens</title><idno type="ISSN">1553-7366</idno><idno type="eISSN">1553-7374</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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 <italic>Staphylococcus aureus</italic> (MRSA) and Vancomycin Resistant <italic>Enterococcus</italic> (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 <italic>Clostridium difficile</italic> also falls within this category. Since 2000, there has been a striking increase in <italic>C. difficile</italic> 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 <italic>C. difficile</italic>. 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 <italic>C. difficile</italic> isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for <italic>C. difficile</italic> pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Borriello, Sp" uniqKey="Borriello S">SP Borriello</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Warny, M" uniqKey="Warny M">M Warny</name></author><author><name sortKey="Pepin, J" uniqKey="Pepin J">J Pepin</name></author><author><name sortKey="Fang, A" uniqKey="Fang A">A Fang</name></author><author><name sortKey="Killgore, G" uniqKey="Killgore G">G Killgore</name></author><author><name sortKey="Thompson, A" uniqKey="Thompson A">A Thompson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcdonald, Lc" uniqKey="Mcdonald L">LC McDonald</name></author><author><name sortKey="Killgore, Ge" uniqKey="Killgore G">GE Killgore</name></author><author><name sortKey="Thompson, A" uniqKey="Thompson A">A Thompson</name></author><author><name sortKey="Owens, Rc" uniqKey="Owens R">RC Owens</name></author><author><name sortKey="Kazakova, Sv" uniqKey="Kazakova S">SV Kazakova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stabler, Ra" uniqKey="Stabler R">RA Stabler</name></author><author><name sortKey="He, M" uniqKey="He M">M He</name></author><author><name sortKey="Dawson, L" uniqKey="Dawson L">L Dawson</name></author><author><name sortKey="Martin, M" uniqKey="Martin M">M Martin</name></author><author><name sortKey="Valiente, E" uniqKey="Valiente E">E Valiente</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Loo, Vg" uniqKey="Loo V">VG Loo</name></author><author><name sortKey="Poirier, L" uniqKey="Poirier L">L Poirier</name></author><author><name sortKey="Miller, Ma" uniqKey="Miller M">MA Miller</name></author><author><name sortKey="Oughton, M" uniqKey="Oughton M">M Oughton</name></author><author><name sortKey="Libman, Md" uniqKey="Libman M">MD Libman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Muto, Ca" uniqKey="Muto C">CA Muto</name></author><author><name sortKey="Pokrywka, M" uniqKey="Pokrywka M">M Pokrywka</name></author><author><name sortKey="Shutt, K" uniqKey="Shutt K">K Shutt</name></author><author><name sortKey="Mendelsohn, Ab" uniqKey="Mendelsohn A">AB Mendelsohn</name></author><author><name sortKey="Nouri, K" uniqKey="Nouri K">K Nouri</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuijper, Ej" uniqKey="Kuijper E">EJ Kuijper</name></author><author><name sortKey="Coignard, B" uniqKey="Coignard B">B Coignard</name></author><author><name sortKey="Tull, P" uniqKey="Tull P">P Tull</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rouphael, Ng" uniqKey="Rouphael N">NG Rouphael</name></author><author><name sortKey="O Donnell, Ja" uniqKey="O Donnell J">JA O'Donnell</name></author><author><name sortKey="Bhatnagar, J" uniqKey="Bhatnagar J">J Bhatnagar</name></author><author><name sortKey="Lewis, F" uniqKey="Lewis F">F Lewis</name></author><author><name sortKey="Polgreen, Pm" uniqKey="Polgreen P">PM Polgreen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zilberberg, Md" uniqKey="Zilberberg M">MD Zilberberg</name></author><author><name sortKey="Tillotson, Gs" uniqKey="Tillotson G">GS Tillotson</name></author><author><name sortKey="Mcdonald, C" uniqKey="Mcdonald C">C McDonald</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Baker, Ss" uniqKey="Baker S">SS Baker</name></author><author><name sortKey="Faden, H" uniqKey="Faden H">H Faden</name></author><author><name sortKey="Sayej, W" uniqKey="Sayej W">W Sayej</name></author><author><name sortKey="Patel, R" uniqKey="Patel R">R Patel</name></author><author><name sortKey="Baker, Rd" uniqKey="Baker R">RD Baker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Riley, Tv" uniqKey="Riley T">TV Riley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Merrigan, M" uniqKey="Merrigan M">M Merrigan</name></author><author><name sortKey="Venugopal, A" uniqKey="Venugopal A">A Venugopal</name></author><author><name sortKey="Mallozzi, M" uniqKey="Mallozzi M">M Mallozzi</name></author><author><name sortKey="Roxas, B" uniqKey="Roxas B">B Roxas</name></author><author><name sortKey="Viswanathan, Vk" uniqKey="Viswanathan V">VK Viswanathan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sebaihia, M" uniqKey="Sebaihia M">M Sebaihia</name></author><author><name sortKey="Wren, Bw" uniqKey="Wren B">BW Wren</name></author><author><name sortKey="Mullany, P" uniqKey="Mullany P">P Mullany</name></author><author><name sortKey="Fairweather, Nf" uniqKey="Fairweather N">NF Fairweather</name></author><author><name sortKey="Minton, N" uniqKey="Minton N">N Minton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Just, I" uniqKey="Just I">I Just</name></author><author><name sortKey="Selzer, J" uniqKey="Selzer J">J Selzer</name></author><author><name sortKey="Wilm, M" uniqKey="Wilm M">M Wilm</name></author><author><name sortKey="Von Eichel Streiber, C" uniqKey="Von Eichel Streiber C">C von Eichel-Streiber</name></author><author><name sortKey="Mann, M" uniqKey="Mann M">M Mann</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lyras, D" uniqKey="Lyras D">D Lyras</name></author><author><name sortKey="O Connor, Jr" uniqKey="O Connor J">JR O'Connor</name></author><author><name sortKey="Howarth, Pk" uniqKey="Howarth P">PK Howarth</name></author><author><name sortKey="Sambol, Sp" uniqKey="Sambol S">SP Sambol</name></author><author><name sortKey="Carter, Gp" uniqKey="Carter G">GP Carter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuehne, Sa" uniqKey="Kuehne S">SA Kuehne</name></author><author><name sortKey="Cartman, St" uniqKey="Cartman S">ST Cartman</name></author><author><name sortKey="Heap, Jt" uniqKey="Heap J">JT Heap</name></author><author><name sortKey="Kelly, Ml" uniqKey="Kelly M">ML Kelly</name></author><author><name sortKey="Cockayne, A" uniqKey="Cockayne A">A Cockayne</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Braun, V" uniqKey="Braun V">V Braun</name></author><author><name sortKey="Hundsberger, T" uniqKey="Hundsberger T">T Hundsberger</name></author><author><name sortKey="Leukel, P" uniqKey="Leukel P">P Leukel</name></author><author><name sortKey="Sauerborn, M" uniqKey="Sauerborn M">M Sauerborn</name></author><author><name sortKey="Eichel Streiber, Cv" uniqKey="Eichel Streiber C">Cv Eichel-Streiber</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mani, N" uniqKey="Mani N">N Mani</name></author><author><name sortKey="Dupuy, B" uniqKey="Dupuy B">B Dupuy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tan, Ks" uniqKey="Tan K">KS Tan</name></author><author><name sortKey="Wee, By" uniqKey="Wee B">BY Wee</name></author><author><name sortKey="Song, Kp" uniqKey="Song K">KP Song</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Matamouros, S" uniqKey="Matamouros S">S Matamouros</name></author><author><name sortKey="England, P" uniqKey="England P">P England</name></author><author><name sortKey="Dupuy, B" uniqKey="Dupuy B">B Dupuy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dupuy, B" uniqKey="Dupuy B">B Dupuy</name></author><author><name sortKey="Govind, R" uniqKey="Govind R">R Govind</name></author><author><name sortKey="Antunes, A" uniqKey="Antunes A">A Antunes</name></author><author><name sortKey="Matamouros, S" uniqKey="Matamouros S">S Matamouros</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Murray, R" uniqKey="Murray R">R Murray</name></author><author><name sortKey="Boyd, D" uniqKey="Boyd D">D Boyd</name></author><author><name sortKey="Levett, Pn" uniqKey="Levett P">PN Levett</name></author><author><name sortKey="Mulvey, Mr" uniqKey="Mulvey M">MR Mulvey</name></author><author><name sortKey="Alfa, Mj" uniqKey="Alfa M">MJ Alfa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mani, N" uniqKey="Mani N">N Mani</name></author><author><name sortKey="Lyras, D" uniqKey="Lyras D">D Lyras</name></author><author><name sortKey="Barroso, L" uniqKey="Barroso L">L Barroso</name></author><author><name sortKey="Howarth, P" uniqKey="Howarth P">P Howarth</name></author><author><name sortKey="Wilkins, T" uniqKey="Wilkins T">T Wilkins</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="O Connor, Jr" uniqKey="O Connor J">JR O'Connor</name></author><author><name sortKey="Lyras, D" uniqKey="Lyras D">D Lyras</name></author><author><name sortKey="Farrow, Ka" uniqKey="Farrow K">KA Farrow</name></author><author><name sortKey="Adams, V" uniqKey="Adams V">V Adams</name></author><author><name sortKey="Powell, Dr" uniqKey="Powell D">DR Powell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Burns, Da" uniqKey="Burns D">DA Burns</name></author><author><name sortKey="Heap, Jt" uniqKey="Heap J">JT Heap</name></author><author><name sortKey="Minton, Np" uniqKey="Minton N">NP Minton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cartman, St" uniqKey="Cartman S">ST Cartman</name></author><author><name sortKey="Minton, Np" uniqKey="Minton N">NP Minton</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Heap, Jt" uniqKey="Heap J">JT Heap</name></author><author><name sortKey="Kuehne, Sa" uniqKey="Kuehne S">SA Kuehne</name></author><author><name sortKey="Ehsaan, M" uniqKey="Ehsaan M">M Ehsaan</name></author><author><name sortKey="Cartman, St" uniqKey="Cartman S">ST Cartman</name></author><author><name sortKey="Cooksley, Cm" uniqKey="Cooksley C">CM Cooksley</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nallapareddy, Sr" uniqKey="Nallapareddy S">SR Nallapareddy</name></author><author><name sortKey="Singh, Kv" uniqKey="Singh K">KV Singh</name></author><author><name sortKey="Murray, Be" uniqKey="Murray B">BE Murray</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Carter, Gp" uniqKey="Carter G">GP Carter</name></author><author><name sortKey="Lyras, D" uniqKey="Lyras D">D Lyras</name></author><author><name sortKey="Allen, Dl" uniqKey="Allen D">DL Allen</name></author><author><name sortKey="Mackin, Ke" uniqKey="Mackin K">KE Mackin</name></author><author><name sortKey="Howarth, Pm" uniqKey="Howarth P">PM Howarth</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Awad, Mm" uniqKey="Awad M">MM Awad</name></author><author><name sortKey="Rood, Ji" uniqKey="Rood J">JI Rood</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hundsberger, T" uniqKey="Hundsberger T">T Hundsberger</name></author><author><name sortKey="Braun, V" uniqKey="Braun V">V Braun</name></author><author><name sortKey="Weidmann, M" uniqKey="Weidmann M">M Weidmann</name></author><author><name sortKey="Leukel, P" uniqKey="Leukel P">P Leukel</name></author><author><name sortKey="Sauerborn, M" uniqKey="Sauerborn M">M Sauerborn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Razaq, N" uniqKey="Razaq N">N Razaq</name></author><author><name sortKey="Sambol, S" uniqKey="Sambol S">S Sambol</name></author><author><name sortKey="Nagaro, K" uniqKey="Nagaro K">K Nagaro</name></author><author><name sortKey="Zukowski, W" uniqKey="Zukowski W">W Zukowski</name></author><author><name sortKey="Cheknis, A" uniqKey="Cheknis A">A Cheknis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goulding, D" uniqKey="Goulding D">D Goulding</name></author><author><name sortKey="Thompson, H" uniqKey="Thompson H">H Thompson</name></author><author><name sortKey="Emerson, J" uniqKey="Emerson J">J Emerson</name></author><author><name sortKey="Fairweather, Nf" uniqKey="Fairweather N">NF Fairweather</name></author><author><name sortKey="Dougan, G" uniqKey="Dougan G">G Dougan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="He, M" uniqKey="He M">M He</name></author><author><name sortKey="Sebaihia, M" uniqKey="Sebaihia M">M Sebaihia</name></author><author><name sortKey="Lawley, Td" uniqKey="Lawley T">TD Lawley</name></author><author><name sortKey="Stabler, Ra" uniqKey="Stabler R">RA Stabler</name></author><author><name sortKey="Dawson, Lf" uniqKey="Dawson L">LF Dawson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rupnik, M" uniqKey="Rupnik M">M Rupnik</name></author><author><name sortKey="Avesani, V" uniqKey="Avesani V">V Avesani</name></author><author><name sortKey="Janc, M" uniqKey="Janc M">M Janc</name></author><author><name sortKey="Von Eichel Streiber, C" uniqKey="Von Eichel Streiber C">C von Eichel-Streiber</name></author><author><name sortKey="Delmee, M" uniqKey="Delmee M">M Delmee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Richards, M" uniqKey="Richards M">M Richards</name></author><author><name sortKey="Knox, J" uniqKey="Knox J">J Knox</name></author><author><name sortKey="Elliott, B" uniqKey="Elliott B">B Elliott</name></author><author><name sortKey="Mackin, K" uniqKey="Mackin K">K Mackin</name></author><author><name sortKey="Lyras, D" uniqKey="Lyras D">D Lyras</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Curry, Sr" uniqKey="Curry S">SR Curry</name></author><author><name sortKey="Marsh, Jw" uniqKey="Marsh J">JW Marsh</name></author><author><name sortKey="Muto, Ca" uniqKey="Muto C">CA Muto</name></author><author><name sortKey="O Leary, Mm" uniqKey="O Leary M">MM O'Leary</name></author><author><name sortKey="Pasculle, Aw" uniqKey="Pasculle A">AW Pasculle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lyerly, Dm" uniqKey="Lyerly D">DM Lyerly</name></author><author><name sortKey="Barroso, La" uniqKey="Barroso L">LA Barroso</name></author><author><name sortKey="Wilkins, Td" uniqKey="Wilkins T">TD Wilkins</name></author><author><name sortKey="Depitre, C" uniqKey="Depitre C">C Depitre</name></author><author><name sortKey="Corthier, G" uniqKey="Corthier G">G Corthier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Carter, Gp" uniqKey="Carter G">GP Carter</name></author><author><name sortKey="Rood, Ji" uniqKey="Rood J">JI Rood</name></author><author><name sortKey="Lyras, D" uniqKey="Lyras D">D Lyras</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="O Connor, Jr" uniqKey="O Connor J">JR O'Connor</name></author><author><name sortKey="Johnson, S" uniqKey="Johnson S">S Johnson</name></author><author><name sortKey="Gerding, Dn" uniqKey="Gerding D">DN Gerding</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Heap, Jt" uniqKey="Heap J">JT Heap</name></author><author><name sortKey="Pennington, Oj" uniqKey="Pennington O">OJ Pennington</name></author><author><name sortKey="Cartman, St" uniqKey="Cartman S">ST Cartman</name></author><author><name sortKey="Carter, Gp" uniqKey="Carter G">GP Carter</name></author><author><name sortKey="Np, M" uniqKey="Np M">M NP.</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mullany, P" uniqKey="Mullany P">P Mullany</name></author><author><name sortKey="Wilks, M" uniqKey="Wilks M">M Wilks</name></author><author><name sortKey="Tabaqchali, S" uniqKey="Tabaqchali S">S Tabaqchali</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Minton, N" uniqKey="Minton N">N Minton</name></author><author><name sortKey="Carter, G" uniqKey="Carter G">G Carter</name></author><author><name sortKey="Herbert, M" uniqKey="Herbert M">M Herbert</name></author><author><name sortKey="O Keeffe, T" uniqKey="O Keeffe T">T O'Keeffe</name></author><author><name sortKey="Purdy, D" uniqKey="Purdy D">D Purdy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Goorhuis, A" uniqKey="Goorhuis A">A Goorhuis</name></author><author><name sortKey="Debast, Sb" uniqKey="Debast S">SB Debast</name></author><author><name sortKey="Van Leengoed, La" uniqKey="Van Leengoed L">LA van Leengoed</name></author><author><name sortKey="Harmanus, C" uniqKey="Harmanus C">C Harmanus</name></author><author><name sortKey="Notermans, Dw" uniqKey="Notermans D">DW Notermans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dawson, Lf" uniqKey="Dawson L">LF Dawson</name></author><author><name sortKey="Valiente, E" uniqKey="Valiente E">E Valiente</name></author><author><name sortKey="Wren, Bw" uniqKey="Wren B">BW Wren</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stabler, Ra" uniqKey="Stabler R">RA Stabler</name></author><author><name sortKey="Dawson, Lf" uniqKey="Dawson L">LF Dawson</name></author><author><name sortKey="Phua, Lt" uniqKey="Phua L">LT Phua</name></author><author><name sortKey="Wren, Bw" uniqKey="Wren B">BW Wren</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lanis, Jm" uniqKey="Lanis J">JM Lanis</name></author><author><name sortKey="Barua, S" uniqKey="Barua S">S Barua</name></author><author><name sortKey="Ballard, Jd" uniqKey="Ballard J">JD Ballard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Schwan, C" uniqKey="Schwan C">C Schwan</name></author><author><name sortKey="Stecher, B" uniqKey="Stecher B">B Stecher</name></author><author><name sortKey="Tzivelekidis, T" uniqKey="Tzivelekidis T">T Tzivelekidis</name></author><author><name sortKey="Van Ham, M" uniqKey="Van Ham M">M van Ham</name></author><author><name sortKey="Rohde, M" uniqKey="Rohde M">M Rohde</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Akerlund, T" uniqKey="Akerlund T">T Akerlund</name></author><author><name sortKey="Persson, I" uniqKey="Persson I">I Persson</name></author><author><name sortKey="Unemo, M" uniqKey="Unemo M">M Unemo</name></author><author><name sortKey="Noren, T" uniqKey="Noren T">T Noren</name></author><author><name sortKey="Svenungsson, B" uniqKey="Svenungsson B">B Svenungsson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lawley, Td" uniqKey="Lawley T">TD Lawley</name></author><author><name sortKey="Clare, S" uniqKey="Clare S">S Clare</name></author><author><name sortKey="Walker, Aw" uniqKey="Walker A">AW Walker</name></author><author><name sortKey="Goulding, D" uniqKey="Goulding D">D Goulding</name></author><author><name sortKey="Stabler, Ra" uniqKey="Stabler R">RA Stabler</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Johnson, S" uniqKey="Johnson S">S Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lindsay, Ja" uniqKey="Lindsay J">JA Lindsay</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bonten, Mj" uniqKey="Bonten M">MJ Bonten</name></author><author><name sortKey="Willems, R" uniqKey="Willems R">R Willems</name></author><author><name sortKey="Weinstein, Ra" uniqKey="Weinstein R">RA Weinstein</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Smith, Cj" uniqKey="Smith C">CJ Smith</name></author><author><name sortKey="Markowitz, Sm" uniqKey="Markowitz S">SM Markowitz</name></author><author><name sortKey="Macrina, Fl" uniqKey="Macrina F">FL Macrina</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sambrook, J" uniqKey="Sambrook J">J Sambrook</name></author><author><name sortKey="Russell, Dw" uniqKey="Russell D">DW Russell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Awad, Mm" uniqKey="Awad M">MM Awad</name></author><author><name sortKey="Bryant, Ae" uniqKey="Bryant A">AE Bryant</name></author><author><name sortKey="Stevens, Dl" uniqKey="Stevens D">DL Stevens</name></author><author><name sortKey="Rood, Ji" uniqKey="Rood J">JI Rood</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Govind, R" uniqKey="Govind R">R Govind</name></author><author><name sortKey="Vediyappan, G" uniqKey="Vediyappan G">G Vediyappan</name></author><author><name sortKey="Rolfe, Rd" uniqKey="Rolfe R">RD Rolfe</name></author><author><name sortKey="Fralick, Ja" uniqKey="Fralick J">JA Fralick</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Abramoff, Md" uniqKey="Abramoff M">MD Abramoff</name></author><author><name sortKey="Magelhaes, Pj" uniqKey="Magelhaes P">PJ Magelhaes</name></author><author><name sortKey="Ram, Sj" uniqKey="Ram S">SJ Ram</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Marsh, Jw" uniqKey="Marsh J">JW Marsh</name></author><author><name sortKey="O Leary, Mm" uniqKey="O Leary M">MM O'Leary</name></author><author><name sortKey="Shutt, Ka" uniqKey="Shutt K">KA Shutt</name></author><author><name sortKey="Pasculle, Aw" uniqKey="Pasculle A">AW Pasculle</name></author><author><name sortKey="Johnson, S" uniqKey="Johnson S">S Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Boyer, Hw" uniqKey="Boyer H">HW Boyer</name></author><author><name sortKey="Roulland Dussoix, D" uniqKey="Roulland Dussoix D">D Roulland-Dussoix</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wust, J" uniqKey="Wust J">J Wust</name></author><author><name sortKey="Sullivan, Nm" uniqKey="Sullivan N">NM Sullivan</name></author><author><name sortKey="Hardegger, U" uniqKey="Hardegger U">U Hardegger</name></author><author><name sortKey="Wilkins, Td" uniqKey="Wilkins T">TD Wilkins</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mullany, P" uniqKey="Mullany P">P Mullany</name></author><author><name sortKey="Wilks, M" uniqKey="Wilks M">M Wilks</name></author><author><name sortKey="Lamb, I" uniqKey="Lamb I">I Lamb</name></author><author><name sortKey="Clayton, C" uniqKey="Clayton C">C Clayton</name></author><author><name sortKey="Wren, B" uniqKey="Wren B">B Wren</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS Pathog</journal-id><journal-id journal-id-type="iso-abbrev">PLoS Pathog</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plospath</journal-id><journal-title-group><journal-title>PLoS Pathogens</journal-title></journal-title-group><issn pub-type="ppub">1553-7366</issn><issn pub-type="epub">1553-7374</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22022270</article-id><article-id pub-id-type="pmc">3192846</article-id><article-id pub-id-type="publisher-id">PPATHOGENS-D-11-00972</article-id><article-id pub-id-type="doi">10.1371/journal.ppat.1002317</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biology</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Medicine</subject></subj-group></article-categories><title-group><article-title>The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of <italic>Clostridium difficile</italic></article-title><alt-title alt-title-type="running-head">TcdC Modulates Hypervirulence in <italic>C. difficile</italic></alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Carter</surname><given-names>Glen P.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Douce</surname><given-names>Gillian R.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Govind</surname><given-names>Revathi</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Howarth</surname><given-names>Pauline M.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Mackin</surname><given-names>Kate E.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Spencer</surname><given-names>Janice</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Buckley</surname><given-names>Anthony M.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Antunes</surname><given-names>Ana</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kotsanas</surname><given-names>Despina</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Jenkin</surname><given-names>Grant A.</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author"><name><surname>Dupuy</surname><given-names>Bruno</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Rood</surname><given-names>Julian I.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lyras</surname><given-names>Dena</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Microbiology, Monash University, Clayton, Victoria, Australia</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Division of Infection and Immunity, FBLS Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Division of Biology, Kansas State University, Manhattan, Kansas, United States of America</addr-line></aff><aff id="aff4"><label>4</label><addr-line>Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France</addr-line></aff><aff id="aff5"><label>5</label><addr-line>Department of Infectious Diseases, Southern Health, Monash Medical Centre, Clayton, Victoria, Australia</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Koehler</surname><given-names>Theresa M.</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">The University of Texas-Houston Medical School, United States of America</aff><author-notes><corresp id="cor1">* E-mail: <email>Dena.Lyras@monash.edu</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: GPC BD GAJ JIR GRD DL. Performed the experiments: GPC PMH KEM DK RG GRD JS AMB AA. Analyzed the data: GPC BD GAJ JIR PMH KEM DK RG AA GRD JS AMB DL. Contributed reagents/materials/analysis tools: GPC BD GAJ JIR DK RG GRD DL. Wrote the paper: GPC DL.</p></fn></author-notes><pub-date pub-type="collection"><month>10</month><year>2011</year></pub-date><pub-date pub-type="epub"><day>13</day><month>10</month><year>2011</year></pub-date><volume>7</volume><issue>10</issue><elocation-id>e1002317</elocation-id><history><date date-type="received"><day>10</day><month>5</month><year>2011</year></date><date date-type="accepted"><day>30</day><month>8</month><year>2011</year></date></history><permissions><copyright-statement>Carter et al.</copyright-statement><copyright-year>2011</copyright-year><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</license-p></license></permissions><abstract><p>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 <italic>Staphylococcus aureus</italic> (MRSA) and Vancomycin Resistant <italic>Enterococcus</italic> (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 <italic>Clostridium difficile</italic> also falls within this category. Since 2000, there has been a striking increase in <italic>C. difficile</italic> 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 <italic>C. difficile</italic>. 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 <italic>C. difficile</italic> isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for <italic>C. difficile</italic> pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.</p></abstract><abstract abstract-type="summary"><title>Author Summary</title><p>Hospital infections are increasingly being recognised as a major patient safety issue with the hospital environment providing a niche for the rapid evolution of microbial pathogens that are well adapted to infecting susceptible patients. The spore-forming <italic>Clostridium difficile</italic> is one such bacterium, which causes disease in patients undergoing antibiotic therapy. Since 2000, there has been a striking increase in <italic>C. difficile</italic> infections due to the emergence of hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Here we use a genetic approach to identify a factor linked to the development of hypervirulence in <italic>C. difficile</italic>. This study shows that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor contributing to the development of hypervirulence in epidemic isolates, presumably because it leads to significantly increased toxin production. These results have important implications for <italic>C. difficile</italic> pathogenesis since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype. This study has increased our understanding of how these new variant strains cause disease and why they are more harmful, which is critical for the development of improved strategies for preventing and treating these infections.</p></abstract><counts><page-count count="11"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p><italic>C. difficile</italic> is the causative agent of a spectrum of gastrointestinal diseases, collectively known as <italic>C. difficile</italic> infections, or CDI, that are induced by treatment with antibiotics that disrupt the normal gastrointestinal microbiota. CDI can range from mild diarrhoea, through moderately serious disease, to severe life-threatening pseudomembranous colitis, a chronic, often fatal, gastrointestinal disease <xref rid="ppat.1002317-Borriello1" ref-type="bibr">[1]</xref>. During the past decade, there has been an astonishing increase in the rate and prevalence of <italic>C. difficile</italic> infections in many parts of the world, including the UK, USA, Canada and Europe, largely due to the emergence of a “hypervirulent” or epidemic group of isolates belonging to the BI/NAP1/027 category <xref rid="ppat.1002317-Warny1" ref-type="bibr">[2]</xref>, <xref rid="ppat.1002317-McDonald1" ref-type="bibr">[3]</xref>. These strains are highly resistant to fluoroquinolones <xref rid="ppat.1002317-McDonald1" ref-type="bibr">[3]</xref> and are associated with more severe disease and higher mortality rates <xref rid="ppat.1002317-Stabler1" ref-type="bibr">[4]</xref>–<xref rid="ppat.1002317-Kuijper1" ref-type="bibr">[7]</xref>. <italic>C. difficile</italic> now also causes disease in those previously not at risk, such as children and pregnant women, with community-associated <italic>C. difficile</italic> disease being increasingly common <xref rid="ppat.1002317-Rouphael1" ref-type="bibr">[8]</xref>–<xref rid="ppat.1002317-Baker1" ref-type="bibr">[10]</xref>.</p><p>The reasons for the emergence of these strains, and for their increased virulence, remain largely speculative. The use of fluoroquinolones, and the emergence of fluoroquinolone resistant strains, are undoubtedly driving factors in these new epidemics <xref rid="ppat.1002317-Riley1" ref-type="bibr">[11]</xref>, however, the reasons for the heightened virulence and persistence of these strains are unknown. Genotypic and phenotypic comparison of the hypervirulent BI/NAP1/027 isolates to historical strains has identified numerous differences that may contribute to hypervirulence. Phenotypically, these differences may include the production of a toxin known as binary toxin, or CDT <xref rid="ppat.1002317-McDonald1" ref-type="bibr">[3]</xref>, and a higher sporulation rate <xref rid="ppat.1002317-Merrigan1" ref-type="bibr">[12]</xref>. Whole genome comparisons have identified numerous genetic differences with BI/NAP1/027 strains having an additional 234 genes compared to the well characterised strain 630 <xref rid="ppat.1002317-Sebaihia1" ref-type="bibr">[13]</xref>, including five unique genetic regions that are absent from both strain 630 and non-epidemic 027 strains <xref rid="ppat.1002317-Stabler1" ref-type="bibr">[4]</xref>. Fundamentally, however, the factors directly resulting in the development of hypervirulence by these strains remain unknown.</p><p>The major virulence factors of <italic>C. difficile</italic> are two members of the large clostridial cytotoxin family, toxin A and toxin B, encoded by the <italic>tcdA</italic> and <italic>tcdB</italic> genes, respectively, which are potent monoglucosyltransferases that irreversibly modify members of the Rho family of host regulatory proteins <xref rid="ppat.1002317-Just1" ref-type="bibr">[14]</xref>. Two recent studies definitively showed that toxin B plays a major role in the virulence of <italic>C. difficile</italic><xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>, <xref rid="ppat.1002317-Kuehne1" ref-type="bibr">[16]</xref>. The role of toxin A in disease was less clear however, with conflicting data concerning toxin A reported <xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>, <xref rid="ppat.1002317-Kuehne1" ref-type="bibr">[16]</xref>.</p><p>Epidemic strains are reported to produce significantly more toxin A and toxin B than other strains <xref rid="ppat.1002317-Warny1" ref-type="bibr">[2]</xref>. The <italic>tcdA</italic> and <italic>tcdB</italic> genes are located on the chromosome within a region known as the pathogenicity locus or PaLoc <xref rid="ppat.1002317-Braun1" ref-type="bibr">[17]</xref>. In addition to <italic>tcdA</italic> and <italic>tcdB</italic>, the PaLoc encodes three additional genes designated <italic>tcdR</italic>, <italic>tcdE</italic> and <italic>tcdC</italic>, which encode an alternative sigma factor, TcdR <xref rid="ppat.1002317-Mani1" ref-type="bibr">[18]</xref>, a putative holin, TcdE <xref rid="ppat.1002317-Tan1" ref-type="bibr">[19]</xref>, and an anti-sigma factor, TcdC <xref rid="ppat.1002317-Matamouros1" ref-type="bibr">[20]</xref>, respectively. The expression of toxins A and B is controlled in a complex manner by several factors, including TcdR and TcdC. TcdC is thought to negatively regulate toxin production by interacting with TcdR or with TcdR-containing RNA polymerase holoenzyme or both <xref rid="ppat.1002317-Matamouros1" ref-type="bibr">[20]</xref>, TcdR is essential for toxin production <xref rid="ppat.1002317-Mani1" ref-type="bibr">[18]</xref>. BI/NAP1/027 <italic>C. difficile</italic> strains have a nonsense mutation in <italic>tcdC</italic>, which results in the production of a truncated protein that no longer negatively regulates TcdR. This mutation is postulated to be responsible for the increased toxin production observed <italic>in vitro</italic> in these strains <xref rid="ppat.1002317-Warny1" ref-type="bibr">[2]</xref>. Accordingly, this observation has prompted debate over the importance of the <italic>tcdC</italic> mutation in the hypervirulent phenotype. However, there is currently a lack of experimental evidence to support this hypothesis, with inconsistent reports in the published literature <xref rid="ppat.1002317-Matamouros1" ref-type="bibr">[20]</xref>–<xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>.</p><p>Despite their important impact worldwide on public health little is known about the virulence factors of BI/NAP1/027 strains and many important questions about the pathogenesis of disease caused by these strains remain to be answered, especially the role played by TcdC. BI/NAP1/027 isolates have proven difficult to genetically manipulate, which has hampered our ability to study these strains at the molecular level. To address these questions, here we use a novel Tn<italic>916</italic>-based plasmid conjugation system to facilitate the efficient transfer of plasmids into BI/NAP1/027 strains of <italic>C. difficile</italic>. Using this system, we have demonstrated conclusively the role of TcdC as a negative regulator of toxin production in <italic>C. difficile</italic>. Furthermore, using the hamster model of infection, we provide evidence to show that the <italic>tcdC</italic> mutation found in BI/NAP1/027 strains is an important factor in the development of hypervirulence by these strains. This study is the first to use isogenic strains to identify a factor involved in the development of a hypervirulent phenotype in <italic>C. difficile</italic>, and also represents the first <italic>in vivo</italic> demonstration of the role of TcdC in the pathogenesis of <italic>C. difficile</italic> disease.</p></sec><sec id="s2"><title>Results</title><sec id="s2a"><title>Complementation of the <italic>tcdC</italic> mutation in a BI/NAP1/027 epidemic isolate <italic>in trans</italic></title><p>To determine if mutation of the <italic>tcdC</italic> gene in <italic>C. difficile</italic> BI/NAP1/027 isolates leads to the development of a hypervirulent phenotype it was necessary to construct isogenic BI/NAP1/027 strains that only differed in their ability to produce a functional TcdC protein. To construct the isogenic strains required for this analysis, genetic manipulation of BI/NAP1/027 isolates was required. The genetic manipulation of these strains has proved difficult and attempts to transfer plasmids into BI/NAP1/027 strains using published methods, which rely on RP4-mediated conjugation from <italic>Escherichia coli</italic><xref rid="ppat.1002317-Mani2" ref-type="bibr">[23]</xref>–<xref rid="ppat.1002317-Heap1" ref-type="bibr">[27]</xref>, were not successful, even though transfer of plasmids into the genetically amenable strains JIR8094, an erythromycin sensitive derivative of strain 630 <xref rid="ppat.1002317-OConnor1" ref-type="bibr">[24]</xref>, and CD37 was readily achieved (<xref ref-type="supplementary-material" rid="ppat.1002317.s001">Table S1</xref>). To overcome this barrier and to facilitate DNA transfer into the strains of interest, we developed a novel plasmid transfer system that exploits the conjugation apparatus encoded by the broad-host range transposon Tn<italic>916</italic>.</p><p>The <italic>oriT</italic> region of Tn<italic>916</italic> (<italic>oriT</italic><sub>Tn<italic>916</italic></sub>) <xref rid="ppat.1002317-Nallapareddy1" ref-type="bibr">[28]</xref> was cloned into the <italic>catP</italic>-containing <italic>C. difficile</italic> shuttle plasmid pMTL9361Cm <xref rid="ppat.1002317-Carter1" ref-type="bibr">[29]</xref>, generating pDLL4. This plasmid was introduced into <italic>C. perfringens</italic> strain JIR4225, which contains five copies of Tn<italic>916</italic><xref rid="ppat.1002317-Awad1" ref-type="bibr">[30]</xref> and plate matings were performed between this donor strain and several <italic>C. difficile</italic> strains, including a BI/NAP1/027 strain, M7404, which is a Canadian epidemic isolate <xref rid="ppat.1002317-Carter1" ref-type="bibr">[29]</xref>. Transconjugants from these matings were isolated on medium supplemented with thiamphenicol and cefoxitin. The efficiency of plasmid transfer into strain M7404 was 1.2×10<sup>2</sup>–4×10<sup>4</sup> transconjugants/ml of plated culture. Analysis of transconjugants using PCR specific for the <italic>catP</italic> gene together with restriction analysis confirmed that all putative colonies carried pDLL4 (data not shown), verifying successful plasmid transfer into the BI/NAP1/027 strain M7404. Similar plasmid transfer efficiencies were obtained for numerous other <italic>C. difficile</italic> strains (<xref ref-type="supplementary-material" rid="ppat.1002317.s001">Table S1</xref>), highlighting the utility of this methodology for the genetic manipulation of clinically relevant strains.</p><p>To complement the <italic>tcdC</italic> mutation in a BI/NAP1/027 strain the intact <italic>tcdC</italic> gene from strain VPI10463, together with 300 bp of its upstream region, was cloned into the shuttle plasmid pDLL4, generating pDLL17. This plasmid was transferred by Tn<italic>916</italic>-mediated conjugation from <italic>C. perfringens</italic> strain JIR4225 to <italic>C. difficile</italic> strain M7404 as before. PCR was subsequently used to confirm the presence of plasmid pDLL17 in representative transconjugants (data not shown).</p><p>To determine whether the presence of pDLL17 complemented the TcdC deficiency of M7404, Western immunoblots using TcdC-specific antibodies were performed. Lysates were collected from the wild-type M7404, the pDLL4-carrying vector control strain M7404(VC) and the pDLL17-<italic>tcdC</italic><sup>+</sup> strain M7404(<italic>tcdC</italic><sup>+</sup>), as well as strains VPI10463 and the PaLoc-deficient strain VPI11186, which served as positive and negative controls, respectively. An additional control strain, M7404(cured), was generated by serially passaging strain M7404(<italic>tcdC</italic><sup>+</sup>) on non-selective growth medium and curing the plasmid from this strain. Loss of the plasmid was confirmed by sensitivity of the strain to thiamphenicol followed by PCR analysis to verify the absence of several plasmid encoded genes (data not shown). As <xref ref-type="fig" rid="ppat-1002317-g001">Figure 1A</xref> shows, whilst no TcdC could be detected in the lysates of the negative control strain, the wild-type M7404, M7404(VC) and the plasmid-cured strain M7404(cured), a 34-kDa protein that reacted with TcdC-specific antibodies was detected in lysates from the <italic>tcdC</italic><sup>+</sup>-complemented strain M7404(<italic>tcdC</italic><sup>+</sup>). This band was the same size as the immunoreactive TcdC protein produced by the positive control strain VPI10463, confirming that the <italic>tcdC</italic> mutation in the BI/NAP1/027 epidemic isolate M7404 was efficiently complemented <italic>in trans</italic>. Since complementation was performed using a multicopy plasmid, we also quantified TcdC production levels from strain M7404(<italic>tcdC</italic><sup>+</sup>) in comparison to strain VPI10463 using a time-course assay. Previous studies involving transcriptional analysis of PaLoc genes during different growth phases showed that <italic>tcdC</italic> is expressed in early exponential phase but not in stationary phase, whereas the other PaLoc genes show the opposite expression pattern <xref rid="ppat.1002317-Hundsberger1" ref-type="bibr">[31]</xref>. VPI10463 (<xref ref-type="fig" rid="ppat-1002317-g001">Figure 1B</xref>) and M7404(<italic>tcdC</italic><sup>+</sup>) (<xref ref-type="fig" rid="ppat-1002317-g001">Figure 1C</xref>) exhibited similar TcdC expression patterns, with higher levels of TcdC observed in early exponential phase and negligible amounts detected beyond 16 hours, suggesting that the regulatory regions governing <italic>tcdC</italic> expression have been retained on the <italic>tcdC</italic>-carrying fragment used to construct pDLL17. In addition to the kinetics of TcdC expression in strain M7404(<italic>tcdC</italic><sup>+</sup>) mirroring that of VPI10463, a similar amount of protein was also detected at each time point with VPI10463 producing 1.3- to 1.6-fold more protein (<xref ref-type="fig" rid="ppat-1002317-g001">Figure 1B</xref>) than M7404(<italic>tcdC</italic><sup>+</sup>) (<xref ref-type="fig" rid="ppat-1002317-g001">Figure 1C</xref>). Therefore, although <italic>tcdC</italic> complementation was achieved using a multicopy plasmid vector, a physiologically relevant amount of TcdC protein was expressed during the appropriate growth phases in strain M7404(<italic>tcdC</italic><sup>+</sup>).</p><fig id="ppat-1002317-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1002317.g001</object-id><label>Figure 1</label><caption><title>Western blot analysis of TcdC production by wild-type and complemented <italic>C. difficile</italic> strains.</title><p>(A) Qualitative analysis of TcdC production. VPI10463 is the positive control strain; VPI11186 is the negative control strain; M7404 is a wild-type Canadian BI/NAP1/027 strain; M7404(VC) is the wild-type strain carrying the shuttle plasmid pDLL4; M7404(<italic>tcdC</italic><sup>+</sup>) is the wild-type strain carrying the <italic>tcdC</italic> expression plasmid pDLL17 and M7404(cured) is the M7404(<italic>tcdC</italic><sup>+</sup>) strain cured of pDLL17. (B) Time course analysis of TcdC production by the positive control strain <italic>C. difficile</italic> VPI10463. Samples were taken at the indicated times shown in hours. 60 ng of purified recombinant his-tagged TcdC protein (rTcdC) was used as the positive reference sample. (C) Time course analysis of TcdC production by <italic>C. difficile</italic> strain M7404(<italic>tcdC</italic><sup>+</sup>). Samples were taken at the indicated times shown in hours. 300 ng of purified recombinant his-tagged TcdC protein (rTcdC) was used as the positive reference sample. Western blots were performed with rabbit TcdC-specific antibodies. Size standards are shown (kDa).</p></caption><graphic xlink:href="ppat.1002317.g001"></graphic></fig></sec><sec id="s2b"><title>TcdC-mediated repression of toxin production in <italic>C. difficile</italic></title><p>To determine the effect of TcdC on toxin production in strain M7404, a combination of Western immunoblots and cytotoxicity assays were performed using supernatants collected from strain M7404 and isogenic M7404 derivatives carrying the vector pDLL4, pDLL17, the cured strain M7404(cured) and the PaLoc-negative control strain CD37. To assess toxin A production, Western immunoblotting was performed using TcdA-specific antibodies (<xref ref-type="fig" rid="ppat-1002317-g002">Figure 2A</xref>). The results showed that the presence of the <italic>tcdC</italic><sup>+</sup> plasmid pDLL17 resulted in a dramatic decrease in the amount of toxin A produced by M7404(<italic>tcdC<sup>+</sup></italic>) when compared to the wild-type strain. By contrast, M7404(cured) produced qualitatively similar levels of toxin A to wild-type, as did M7404 carrying the vector plasmid, whereas the PaLoc-negative strain CD37 produced no detectable toxin A, as expected.</p><fig id="ppat-1002317-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1002317.g002</object-id><label>Figure 2</label><caption><title>Analysis of the effect of TcdC complementation on toxin production and PaLoc gene expression by <italic>C. difficile</italic>.</title><p>CD37 is the negative control strain; M7404 is the wild-type BI/NAPI/027 strain; M7404(<italic>tcdC</italic><sup>+</sup>) is the wild-type strain carrying the <italic>tcdC</italic> expression vector pDLL17; M7404(VC) is the wild-type strain carrying shuttle plasmid pDLL4 and M7404(cured) is the M7404(<italic>tcdC</italic><sup>+</sup>) strain cured of pDLL17. JIR8094 is a derivative of strain 630. (A) Western blot using toxin-A-specific antibodies. Size standards are shown (kDa) (B) Toxin cytotoxicity assays using Vero cells. Strains are as described above. Lined bars represent the wild-type strain M7404; white bars represent M7404(<italic>tcdC</italic><sup>+</sup>); grey bars represent M7404(VC) and black bars represent M7404(cured). Data represent the mean ± s.e.m. (n = 3). (C) Time course of toxin production measured using Vero cell cytotoxicity assays. Strains are as described above and are represented as follows: M7404(<italic>tcdC</italic><sup>+</sup>) (▪), M7404(VC) (•) and JIR8094 (▴). Note that CD37 was included in this analysis but displayed no toxin production; the line representing this strain is therefore not visible. Data represent the mean ± s.e.m. (n = 3). (D) PaLoc gene specific qRT-PCR. Bars correspond to strains as before and PaLoc genes are indicated. Data represent the mean fold-expression ± s.e.m. (n = 3), compared to the M7404(<italic>tcdC</italic><sup>+</sup>) strain.</p></caption><graphic xlink:href="ppat.1002317.g002"></graphic></fig><p>Vero cell cytotoxicity assays, which predominantly measure toxin B activity <xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>, were then performed to quantitatively determine the effect of TcdC on toxin production from these strains. As previously observed with toxin A, the amount of toxin produced from strain M7404 was significantly reduced when functional TcdC was restored (<xref ref-type="fig" rid="ppat-1002317-g002">Figure 2B</xref>). The amount of toxin produced in the TcdC-complemented strain was approximately 16–32-fold less, and therefore significantly lower (p = 0.0001; unpaired t-test, 95% confidence interval), than in the vector control-carrying M7404 derivative. There was, however, no significant difference in toxin activity levels between strains M7404, the vector control strain M7404(VC) or M7404(cured) (<xref ref-type="fig" rid="ppat-1002317-g002">Figure 2B</xref>). A kinetic analysis of toxin production also clearly showed that the presence of TcdC delayed the onset of toxin production in M7404(<italic>tcdC<sup>+</sup></italic>) in comparison to M7404 carrying the vector plasmid, mirroring the delayed toxin production observed from the <italic>tcdC</italic><sup>+</sup> 630-strain derivative JIR8094 (<xref ref-type="fig" rid="ppat-1002317-g002">Figure 2C</xref>).</p><p>We also determined if TcdC-mediated repression of toxin production was at the transcriptional level and evaluated the effect of <italic>tcdC</italic> complementation on the expression of the other PaLoc-encoded genes, <italic>tcdR</italic> and <italic>tcdE</italic>. Quantitative real-time PCR (qRT-PCR) analysis using RNA extracted from the wild-type strain and its isogenic derivatives was performed to ascertain the relative transcription levels of the <italic>tcdA</italic>, <italic>tcdB</italic>, <italic>tcdR</italic> and <italic>tcdE</italic> genes. As shown in <xref ref-type="fig" rid="ppat-1002317-g002">Figure 2D</xref>, an approximate 13- and 23-fold reduction in <italic>tcdA-</italic> and <italic>tcdB-</italic>specific mRNA levels, respectively, in strain M7404(<italic>tcdC</italic><sup>+</sup>) was observed compared to M7404. Similar observations were made for <italic>tcdR</italic> and <italic>tcdE</italic> expression levels, with 33-fold and 21-fold less <italic>tcdR</italic> and <italic>tcdE</italic> mRNA, respectively, in the <italic>tcdC</italic>-complemented strain compared to the wild-type. No significant differences in the expression levels of these four genes were detected when M7404, the vector-carrying derivative or the cured strain were compared. These data conclusively demonstrate that TcdC negatively regulates toxin production in <italic>C. difficile</italic> and show that repression occurs at the transcriptional level.</p></sec><sec id="s2c"><title>Reduction of the virulence of a BI/NAP1/027 isolate <italic>via</italic> complementation with TcdC</title><p>To define the role of TcdC in the virulence of a BI/NAP1/027 <italic>C. difficile</italic> isolate, female Golden Syrian hamsters were infected with spores of strain M7404 carrying either the vector control or the <italic>tcdC</italic><sup>+</sup> plasmid (n = 10 and n = 12, respectively). For comparative purposes, a group of hamsters (n = 14) was also infected with strain 630, a strain previously characterised as being less virulent than other clinical isolates <xref rid="ppat.1002317-Razaq1" ref-type="bibr">[32]</xref>. Following infection, all <italic>C. difficile</italic> strains were found to be equally efficient at colonising the hamsters (data not shown). Infection of colonised hamsters was allowed to proceed and animals were monitored by telemetry. The end point of infection was achieved when the core body temperature of the hamsters dropped to 35°C. This parameter has previously been shown to be a reliable indicator of non-recoverable disease <xref rid="ppat.1002317-Goulding1" ref-type="bibr">[33]</xref>. At this point, the animals were immediately culled for animal ethics reasons. Bacteria were then isolated from the culled hamsters, the bacterial load quantified and isolates subjected to MVLA analysis <xref rid="ppat.1002317-Goulding1" ref-type="bibr">[33]</xref> to confirm that these isolates were the same strain as originally used for infection.</p><p>Hamsters infected with the M7404(<italic>tcdC</italic><sup>+</sup>) derivative showed a significant delay (p = 0.0003; Logrank (Mantel-Cox) test; 95% confidence interval) in the mean time taken to reach non-recoverable disease (2370 minutes or 39.5 hours) in comparison to the vector-carrying M7404 group (M7404(VC)), with a mean time of 1869 minutes or 31.15 hours (<xref ref-type="fig" rid="ppat-1002317-g003">Figure 3</xref>). In one of the hamsters colonised with the M7404(VC) strain, the time taken to reach the end point of infection was substantially longer than the other hamsters in this group (2814 minutes or 46.9 hours). This hamster was shown by statistical analysis (p = 0.0405; Grubbs test; 95% confidence interval) to be an outlier and was therefore excluded from the experimental analysis. Note that statistical significance would be retained upon inclusion of this outlier. Interestingly, whilst the mean time to the end point of infection in the strain 630 group of hamsters (2701 minutes or 45.02 hours) was significantly longer than that of hamsters infected with M7404(VC) (p = 0.0001; Logrank (Mantel-Cox) test; 95% confidence interval), there was no significant difference in the mean time taken to achieve non-recoverable disease in the 630 group compared to the M7404(<italic>tcdC</italic><sup>+</sup>) derivative, indicating that the virulence of the TcdC-complemented strain was equivalent to that of strain 630.</p><fig id="ppat-1002317-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1002317.g003</object-id><label>Figure 3</label><caption><title>Virulence of <italic>C. difficile</italic> wild-type and <italic>tcdC</italic>-complemented strains in hamsters.</title><p>Kaplan-Meier survival curve demonstrating time from infection with <italic>C. difficile</italic> to death. M7404(VC), wild-type M7404 carrying shuttle plasmid pDLL4 (•); M7404(<italic>tcdC</italic><sup>+</sup>), wild-type M7404 carrying <italic>tcdC</italic> expression plasmid pDLL17 (▪); and strain 630, a <italic>C. difficile</italic> isolate with known low virulence (▴). Hamsters were infected intragastrically with 10,000 spores from each strain; M7404(VC) (n = 9), M7404(<italic>tcdC</italic><sup>+</sup>) (n = 12) and strain 630 (n = 14).</p></caption><graphic xlink:href="ppat.1002317.g003"></graphic></fig><p>It is apparent from these virulence experiments that the expression of TcdC in a BI/NAP1/027 isolate has an important effect on virulence, resulting in a significant delay in the time needed to reach non-recoverable disease. These data therefore provide compelling evidence that the naturally occurring mutation of <italic>tcdC</italic> in BI/NAP1/027 isolates is an important factor in the development of a hypervirulent phenotype by these strains.</p></sec><sec id="s2d"><title>TcdC status of clinical isolates does not predict toxin production level</title><p>The results presented here show that a BI/NAP1/027 strain complemented with <italic>tcdC</italic> is not as virulent as its isogenic vector-carrying control, suggesting that any <italic>C. difficile</italic> strain that acquires a null TcdC phenotype has the potential to develop a hypervirulent phenotype. Furthermore, phylogenetic studies have shown <italic>C. difficile</italic> to be a genetically diverse species, with disease-causing isolates seemingly arising from multiple lineages, suggesting that virulence in these strains may have evolved independently <xref rid="ppat.1002317-Stabler1" ref-type="bibr">[4]</xref>, <xref rid="ppat.1002317-He1" ref-type="bibr">[34]</xref>. The <italic>tcdC</italic> status of a diverse group of clinical isolates was therefore determined in parallel to the genetic studies described above. One hundred Australian clinical isolates were initially analysed by toxinotyping, a typing method which categorises strains according to variation in the PaLoc region; BI/NAP1/027 strains belong to toxinotype III <xref rid="ppat.1002317-Rupnik1" ref-type="bibr">[35]</xref>. Approximately 5% of these strains were found to belong to a toxinotype associated with a <italic>tcdC</italic> mutation. PCR and sequence analysis was then used to confirm the presence of <italic>tcdC</italic> mutations in each of these isolates. A single BI/NAP1/027 was identified in this survey, designated strain KI <xref rid="ppat.1002317-Richards1" ref-type="bibr">[36]</xref>. One other strain, DLL3053, was particularly interesting since it belonged to toxinotype group III but was not a BI/NAP1/027 isolate. This strain harboured the well documented single base pair deletion at nucleotide position 117 of <italic>tcdC</italic>, and the 18 base pair in-frame deletion from nucleotide 330 to 347 <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref>. Two strains, DLL3054 and DLL3055, were toxinotype V strains with a nonsense mutation at nucleotide position 184 (C184T) and a 39 base pair deletion from nucleotides 341 to 379 <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref> whereas strain DLL3056 was toxinotype XIV and possessed a nonsense mutation at nucleotide position 191 (C191A) and an in-frame 36 base pair deletion from nucleotide 300 to 336.</p><p>To examine the impact of <italic>tcdC</italic> mutations on toxin production in these clinical isolates, Vero cell cytotoxicity assays were performed. These assays, which predominantly detect toxin B, determine the relative amounts of toxin produced by each strain since the expression of toxins A and B is coordinately regulated <xref rid="ppat.1002317-Mani2" ref-type="bibr">[23]</xref>, <xref rid="ppat.1002317-Hundsberger1" ref-type="bibr">[31]</xref>. Strains JIR8094, a derivative of strain 630 <xref rid="ppat.1002317-OConnor1" ref-type="bibr">[24]</xref>, and VPI10463 <xref rid="ppat.1002317-Lyerly1" ref-type="bibr">[38]</xref>, which both possess intact <italic>tcdC</italic> genes, were used as positive reference controls, and the PaLoc-negative strain CD37 was used as a negative control. Strains were grown in glucose-free medium since glucose has previously been shown to repress toxin production <xref rid="ppat.1002317-Mani2" ref-type="bibr">[23]</xref>. As shown in <xref ref-type="fig" rid="ppat-1002317-g004">Figure 4A</xref>, the relative amount of toxin produced by the <italic>tcdC</italic> clinical isolates varied over approximately a 10-fold range. In agreement with the work of others <xref rid="ppat.1002317-Merrigan1" ref-type="bibr">[12]</xref>, <xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>, these results show that the presence of mutations within <italic>tcdC</italic> was not directly correlated with high level toxin production. Strain DLL3053 for example, did not produce levels of toxin significantly different from that of strain JIR8094, which is considered to be a low toxin producer <xref rid="ppat.1002317-Carter2" ref-type="bibr">[39]</xref>. Of all the isolates with <italic>tcdC</italic> mutations, strain KI <xref rid="ppat.1002317-Richards1" ref-type="bibr">[36]</xref> produced the most toxin, and at significantly (p = 0.0406; unpaired t-test, 95% confidence interval) higher levels that were approximately 10-fold more than strain DLL3053 even though both strains have identical <italic>tcdC</italic> alleles.</p><fig id="ppat-1002317-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1002317.g004</object-id><label>Figure 4</label><caption><title>Comparative analysis of toxin production by naturally occurring <italic>tcdC</italic> clinical isolates.</title><p>Vero cell cytotoxicity assays were used to determine toxin production levels. (A) Strain JIR8094 is a <italic>tcdC</italic><sup>+</sup> control strain, CD37 is a PaLoc-negative control strain and KI is an Australian BI/NAPI/027 isolate <xref rid="ppat.1002317-Richards1" ref-type="bibr">[36]</xref>. All other strains (DLL3053-DLL3056) are clinical isolates carrying naturally occurring <italic>tcdC</italic> mutations, collected from Australian hospitals. (B) Toxin production by the <italic>tcdC</italic><sup>+</sup> control strain VPI10463 is shown on a separate bar chart due to the much higher levels of toxin produced. For comparative purposes strain KI is represented on both bar charts. Data represent the mean±s.e.m. (n = 3).</p></caption><graphic xlink:href="ppat.1002317.g004"></graphic></fig><p>In comparison to the TcdC-positive reference strains, all of the <italic>tcdC</italic>-deficient clinical isolates produced more toxin than strain JIR8094 apart from strain DLL3053 (<xref ref-type="fig" rid="ppat-1002317-g004">Figure 4A</xref>). Conversely, however, all strains produced significantly less toxin than VPI10463 (p = 0.0099–0.0202; unpaired t-test, 95% confidence interval), including the BI/NAP1/027 strain KI. VPI10463 produced over 100-fold more toxin than strains JIR8094 and DLL3053 and over 30-fold more than KI (<xref ref-type="fig" rid="ppat-1002317-g004">Figure 4B</xref>). This observation is in agreement with recently published findings, which showed that VPI10463 produced significantly more toxin than other strains <xref rid="ppat.1002317-Merrigan1" ref-type="bibr">[12]</xref>; however, in that study, strains were grown using glucose-rich BHI medium so differential effects of glucose on toxin production by each strain could not be ruled out. In agreement with other studies <xref rid="ppat.1002317-Merrigan1" ref-type="bibr">[12]</xref>, <xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>, <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref>, our data therefore suggest that the <italic>tcdC</italic>-status alone of <italic>C. difficile</italic> isolates is not an accurate predictor of high-level toxin production.</p></sec></sec><sec id="s3"><title>Discussion</title><p>The hypothesis that the naturally-occurring <italic>tcdC</italic> mutation in epidemic BI/NAP1/027 isolates contributes to hypervirulence is widely accepted, despite a lack of supportive experimental evidence. Indeed, the exact role of TcdC in the pathogenesis of <italic>C. difficile</italic> disease has remained controversial with conflicting findings reported in the literature <xref rid="ppat.1002317-Matamouros1" ref-type="bibr">[20]</xref>–<xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>. As a result, several published studies have suggested that there is a need to assess isogenic <italic>tcdC</italic> strains in order to conclusively determine the role of this gene in the virulence of <italic>C. difficile</italic><xref rid="ppat.1002317-Merrigan1" ref-type="bibr">[12]</xref>, <xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>, <xref rid="ppat.1002317-OConnor2" ref-type="bibr">[40]</xref>. We have now constructed such isogenic strains and compared them in an animal model. The results conclusively show that TcdC negatively regulates toxin production in <italic>C. difficile</italic>. Most importantly, complementation of the <italic>tcdC</italic> mutation in the BI/NAP1/027 epidemic isolate M7404 clearly showed that this mutation is an important factor in the development of hypervirulence by this strain since the genetic complementation of <italic>tcdC</italic> reduced virulence in comparison to the wild-type strain.</p><p>To elucidate the role of TcdC in hypervirulence, it was necessary to construct an isogenic panel of BI/NAP1/027 strains that were identical except for the presence or absence of the wild-type <italic>tcdC</italic> gene. Despite the publication of studies describing the successful transfer of plasmids into the BI/NAP1/027 isolate R20291 <xref rid="ppat.1002317-Burns1" ref-type="bibr">[25]</xref>, <xref rid="ppat.1002317-Cartman1" ref-type="bibr">[26]</xref>, <xref rid="ppat.1002317-Heap2" ref-type="bibr">[41]</xref>, this group of strains has remained difficult to work with at the molecular genetic level. As such, a new system that utilised the conjugation apparatus of Tn<italic>916</italic> was developed in this study and used successfully to genetically manipulate a number of clinically relevant isolates, including a BI/NAP1/027 strain of <italic>C. difficile</italic>. Tn<italic>916</italic> is a broad host-range conjugative transposon that was recently used to transfer plasmids into genetically intractable strains of <italic>Enterococcus faecium</italic><xref rid="ppat.1002317-Nallapareddy1" ref-type="bibr">[28]</xref> and has been shown to transfer into <italic>C. difficile</italic><xref rid="ppat.1002317-Mullany1" ref-type="bibr">[42]</xref>, <xref rid="ppat.1002317-Minton1" ref-type="bibr">[43]</xref>. <italic>C. perfringens</italic> was chosen for use as a donor strain in anticipation that it may be more proficient for the transfer of plasmids into <italic>C. difficile</italic> in comparison to the more distantly related <italic>E. coli</italic>. The addition of <italic>oriT</italic><sub>Tn<italic>916</italic></sub> onto the shuttle vector pMTL9361Cm facilitated the efficient transfer of this plasmid into strain M7404 from a Tn<italic>916</italic>-carrying <italic>C. perfringens</italic> strain. Furthermore, this system has been successfully used to transfer shuttle plasmids into every <italic>C. difficile</italic> isolate tested so far (<xref ref-type="supplementary-material" rid="ppat.1002317.s001">Table S1</xref>). Most importantly, this new technology facilitated the complementation of the <italic>tcdC</italic> mutation in strain M7404 enabling the role of TcdC in the virulence of BI/NAP1/027 strains of <italic>C. difficile</italic> to be investigated.</p><p>Previous <italic>in vitro</italic> studies have shown that TcdC is able to sequester the TcdR sigma factor, preventing its association with core RNA polymerase and blocking toxin gene expression <xref rid="ppat.1002317-Matamouros1" ref-type="bibr">[20]</xref>. These experiments suggested that TcdC was important in the regulation of toxin production by <italic>C. difficile</italic>, but the <italic>in vivo</italic> role of this protein was not determined. Conversely, several studies on <italic>C. difficile</italic> clinical isolates <xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>, <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref> showed that the absence of a functional <italic>tcdC</italic> gene was not an accurate predictor of high level toxin production or increased disease severity, indicating that TcdC may not play an important role in virulence in these strains <xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>, <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref>. The analysis of Australian clinical isolates in the present study is in accordance with these latter studies in that isolates with naturally occurring <italic>tcdC</italic> mutations were found to produce toxin at a range of different levels that were not necessarily high. However, since these strains, and those in the other studies <xref rid="ppat.1002317-Murray1" ref-type="bibr">[22]</xref>, <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref>, are not isogenic it is not possible to draw conclusions about the importance of <italic>tcdC</italic> in the context of toxin yield or virulence. By contrast, the isogenic <italic>tcdC</italic> strains studied here clearly show that TcdC is a negative regulator of toxin production since the <italic>tcdC</italic> complemented BI/NAP1/027 <italic>C. difficile</italic> strain produced significantly less toxin A and B than the non-complemented control strains. The finding that TcdC-status is not correlated with toxin production in clinical isolates highlights the limitation of accurately assigning gene function by studying non-isogenic strains, particularly in a highly heterogeneous species such as <italic>C. difficile</italic>. In this context, it might be of interest to study the function of <italic>tcdC</italic> in isogenic strains generated in a different genetic background such as a ribotype 078 isolate.</p><p>Analysis of PaLoc gene expression by qRT-PCR demonstrated that TcdC exerts regulatory control of toxin production at the transcriptional level, and this is in keeping with its proposed role as an anti-sigma factor <xref rid="ppat.1002317-Matamouros1" ref-type="bibr">[20]</xref>. The observation that the expression of <italic>tcdR</italic> and <italic>tcdE</italic> is reduced in the <italic>tcdC</italic>-complemented strain, together with <italic>tcdA</italic> and <italic>tcdB</italic>, is probably because of autoregulation of <italic>tcdR</italic> since TcdR upregulates its own expression and that of the other PaLoc genes <xref rid="ppat.1002317-Mani2" ref-type="bibr">[23]</xref>.</p><p>The virulence of strain M7404 was reduced upon complementation of <italic>tcdC</italic>, clearly demonstrating that the <italic>tcdC</italic> mutation in BI/NAP1/027 strains has a significant impact on virulence and is likely to be an important factor in the development of hypervirulence by these strains. Surprisingly, the virulence of M7404(<italic>tcdC</italic><sup>+</sup>) was found to be equivalent to that of strain 630, which has been shown in other studies to be reduced in virulence in comparison to other isolates, including three other BI-type strains <xref rid="ppat.1002317-Razaq1" ref-type="bibr">[32]</xref>. These findings have important implications for <italic>C. difficile</italic> virulence since they suggest that strains carrying <italic>tcdC</italic> mutations have the inherent potential to develop hypervirulence. The recent emergence of a new class of hypervirulent strains, ribotype 078 <xref rid="ppat.1002317-Goorhuis1" ref-type="bibr">[44]</xref>, may be one such example. These isolates encode a non-functional TcdC protein <xref rid="ppat.1002317-Curry1" ref-type="bibr">[37]</xref>, produce significantly more toxin than non-epidemic strains, are associated with more severe disease as well as higher rates of mortality and are increasingly being identified as the causative agent of CDI <xref rid="ppat.1002317-Goorhuis1" ref-type="bibr">[44]</xref>, <xref rid="ppat.1002317-Dawson1" ref-type="bibr">[45]</xref>.</p><p>Although these experiments show that TcdC-status alone can modulate virulence it is probable that multiple factors working synergistically are necessary for the development of hypervirulence in the BI/NAP1/027 strains. It is likely that the accumulation of multiple genetic changes in addition to the <italic>tcdC</italic> mutation has enabled BI/NAP1/027 strains to become the predominant disease-causing isolates in numerous countries. Of particular importance might be variations in the functional activity of the encoded toxins since these isolates were recently shown to produce a toxin B that shows variation across the C-terminal receptor binding domain of the protein <xref rid="ppat.1002317-Stabler2" ref-type="bibr">[46]</xref>, resulting in more potent activity across a wider range of cell lines in comparison to toxin B from the historical, non-epidemic strain 630 <xref rid="ppat.1002317-Stabler1" ref-type="bibr">[4]</xref>. Furthermore, using the zebrafish embryo model of intoxication, the BI/NAP1/027 toxin B was recently shown to have pronounced <italic>in vivo</italic> cytotoxic activity in comparison to toxin B from VPI10463, another historical non-epidemic isolate, with greater tissue tropism and more extensive tissue destruction observed <xref rid="ppat.1002317-Lanis1" ref-type="bibr">[47]</xref>. Since toxin B is thought to be one of the major virulence factors of <italic>C. difficile</italic><xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>, <xref rid="ppat.1002317-Kuehne1" ref-type="bibr">[16]</xref> these observations suggest that TcdB variations might play an important role in the hypervirulent phenotype.</p><p>There are other factors that may influence <italic>C. difficile</italic> hypervirulence. The BI/NAP1/027 strains encode an additional toxin known as binary toxin or CDT <xref rid="ppat.1002317-Warny1" ref-type="bibr">[2]</xref>. The role of this toxin in CDI remains to be elucidated but a recent study showed that CDT induces the formation of microtubule-based protrusions on the host cell surface thereby increasing <italic>C. difficile</italic> adherence to epithelial cells. Moreover, intestinal colonisation of gnotobiotic mice with a BI/NAP1/027 <italic>C. difficile</italic> strain was significantly reduced in mice treated with CDT-neutralising antibodies in comparison to control mice <xref rid="ppat.1002317-Schwan1" ref-type="bibr">[48]</xref>. These findings suggest that CDT may be an important colonisation factor, enhancing the ability of BI/NAP1/027 strains to initiate infection as well as causing adjunctive tissue damage during later stages of infection, potentially leading to more severe disease. Many BI/NAP1/027 strains are also more proficient at sporulation than non-epidemic <italic>C. difficile</italic> strains <xref rid="ppat.1002317-Merrigan1" ref-type="bibr">[12]</xref>, <xref rid="ppat.1002317-Akerlund1" ref-type="bibr">[49]</xref>. <italic>C. difficile</italic> spores are highly infectious <xref rid="ppat.1002317-Lawley1" ref-type="bibr">[50]</xref> and play a critical role in the transmission of CDI and perhaps in disease relapse, which is a serious problem in patients with CDI <xref rid="ppat.1002317-Johnson1" ref-type="bibr">[51]</xref>. In this context, enhanced sporulation is ostensibly an important adaptation by BI/NAP1/027 isolates, which would result in larger numbers of spores being shed from infected patients and an increased environmental spore load, ultimately leading to higher transmission rates. Finally, the development of fluoroquinolone resistance, in particular to moxifloxacin and gatifloxacin, is unquestionably a major factor in epidemics caused by BI/NAP1/027 strains <xref rid="ppat.1002317-McDonald1" ref-type="bibr">[3]</xref>, <xref rid="ppat.1002317-Riley1" ref-type="bibr">[11]</xref>. In this regard, the hypothetical co-evolution of enhanced virulence traits and antibiotic resistance in <italic>C. difficile</italic> mirrors trends seen with other significant nosocomial pathogens such as Methicillin Resistant <italic>Staphylococcus aureus</italic> (MRSA) <xref rid="ppat.1002317-Lindsay1" ref-type="bibr">[52]</xref> and Vancomycin Resistant <italic>Enterococcus</italic> (VRE) species <xref rid="ppat.1002317-Bonten1" ref-type="bibr">[53]</xref>. In summary, it is clear that our findings represent an important breakthrough in our understanding of the development of hypervirulence in prevailing <italic>C. difficile</italic> isolates and will provide a significant reference point for future studies on epidemic strains and their control.</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and Methods</title><sec id="s4a"><title>Ethics statement</title><p>This study was carried out in strict accordance with the recommendations in the United Kingdoms Home Office Animals (Scientific Procedures) Act of 1986 which outlines the regulation of the use of laboratory animals for the use of animals in scientific procedures. The experiments described were subject to approval by the University of Glasgow Ethics Committee and by a designated Home Office Inspector (Project Number 60/4218). All experiments were subject to the 3 R consideration (refine, reduce and replace) and all efforts were made to minimize suffering.</p></sec><sec id="s4b"><title>Bacterial strains and growth conditions</title><p>The characteristics and origins of all recombinant strains and plasmids are shown in <xref ref-type="table" rid="ppat-1002317-t001">Table 1</xref> and <xref ref-type="supplementary-material" rid="ppat.1002317.s002">Table S2</xref>, respectively. All bacteriological culture media were obtained from Oxoid. <italic>C. difficile</italic> strains were cultured in BHIS <xref rid="ppat.1002317-Smith1" ref-type="bibr">[54]</xref> or TY medium <xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>, unless otherwise stated, in an atmosphere of 10% H<sub>2</sub>, 10% CO<sub>2</sub>, and 80% N<sub>2</sub> at 37°C in a Coy anaerobic chamber. <italic>Escherichia coli</italic> was cultured in 2×YT medium aerobically at 37°C, with shaking for broth cultures. All antibiotics were purchased from Sigma-Aldrich and were used at the following concentrations: cycloserine (Cs, 250 µg/ml), cefoxitin (Cf, 8 µg/ml), thiamphenicol (Tm, 10 µg/ml) or tetracycline (Tc, 10 µg/ml), chloramphenicol (Cm, 25 µg/ml).</p><table-wrap id="ppat-1002317-t001" position="float"><object-id pub-id-type="doi">10.1371/journal.ppat.1002317.t001</object-id><label>Table 1</label><caption><title>Bacterial strains.</title></caption><alternatives><graphic id="ppat-1002317-t001-1" xlink:href="ppat.1002317.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Strain</td><td align="left" rowspan="1" colspan="1">Characteristics</td><td align="left" rowspan="1" colspan="1">Source/Reference</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><bold><italic>E. coli</italic></bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">DH5α</td><td align="left" rowspan="1" colspan="1">F-φ80Δ <italic>lacZ</italic>dM15Δ(<italic>lacZYA</italic>-<italic>argF</italic>)U169 <italic>endA1recA1 hsdr17</italic>(τ<sub>κ</sub><sup>−</sup>m<sub>κ</sub><sup>+</sup>)<italic>deoR thi-1 supE44 gyrA96 relA1</italic></td><td align="left" rowspan="1" colspan="1">Life Technologies</td></tr><tr><td align="left" rowspan="1" colspan="1">TOP10</td><td align="left" rowspan="1" colspan="1">F- <italic>mcr</italic>A Δ(<italic>mrr-hsd</italic>RMS-<italic>mcr</italic>BC) φ80<italic>lac</italic>ZΔM15 Δ<italic>lac</italic>X74 <italic>nup</italic>G <italic>rec</italic>A1 <italic>ara</italic>D139 Δ(<italic>ara-leu</italic>)7697 <italic>gal</italic>U <italic>gal</italic>K <italic>rps</italic>L(Str<sup>R</sup>) <italic>end</italic>A1 λ<sup>−</sup></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">Invitrogen</td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">HB101</td><td align="left" rowspan="1" colspan="1"><italic>thi</italic>-<italic>1 hsdS20</italic>(r<sub>B</sub><sup>−</sup> m<sub>B</sub><sup>−</sup>) <italic>supE44 recAB ara-14 leuB5 proA2 lacY1 galK rpsL20</italic> (Sm<sup>r</sup>) <italic>xyl-5 mtl-1</italic></td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Boyer1" ref-type="bibr">[60]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>C. perfringens</italic></bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">JIR4225</td><td align="left" rowspan="1" colspan="1"><italic>C. perfringens</italic> strain JIR325 with 5 chromosomal copies of Tn<italic>916</italic></td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Awad1" ref-type="bibr">[30]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1"><bold><italic>C. difficile</italic></bold></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">M7404</td><td align="left" rowspan="1" colspan="1">Canadian BI/NAP1/027 isolate</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Carter1" ref-type="bibr">[29]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">630</td><td align="left" rowspan="1" colspan="1">Wild-type <italic>C. difficile</italic> strain; first <italic>C. difficile</italic> genome available</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Wust1" ref-type="bibr">[61]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">JIR8094</td><td align="left" rowspan="1" colspan="1">Erythromycin sensitive derivative of strain 630</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-OConnor1" ref-type="bibr">[24]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">VPI10463</td><td align="left" rowspan="1" colspan="1">PaLoc-positive <italic>C. difficile</italic> isolate</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Lyerly1" ref-type="bibr">[38]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">VPI11186</td><td align="left" rowspan="1" colspan="1">PaLoc-negative <italic>C. difficile</italic> isolate</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Lyerly1" ref-type="bibr">[38]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">CD37</td><td align="left" rowspan="1" colspan="1">PaLoc-negative <italic>C. difficile</italic> isolate</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Mullany2" ref-type="bibr">[62]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">KI</td><td align="left" rowspan="1" colspan="1">Australian BI/NAP1/027 isolate</td><td align="left" rowspan="1" colspan="1"><xref rid="ppat.1002317-Richards1" ref-type="bibr">[36]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">DLL3053</td><td align="left" rowspan="1" colspan="1">Australian toxinotype III clinical isolate</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">DLL3054</td><td align="left" rowspan="1" colspan="1">Australian toxinotype V clinical isolate</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">DLL3055</td><td align="left" rowspan="1" colspan="1">Australian toxinotype V clinical isolate</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">DLL3056</td><td align="left" rowspan="1" colspan="1">Australian toxinotype XIV clinical isolate</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">M7404(VC)</td><td align="left" rowspan="1" colspan="1">DLL3001 (M7404 carrying shuttle plasmid pDLL4)</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">M7404(<italic>tcdC</italic><sup>+</sup>)</td><td align="left" rowspan="1" colspan="1">DLL3002 (M7404 carrying <italic>tcdC</italic> expression plasmid pDLL17)</td><td align="left" rowspan="1" colspan="1">This study</td></tr><tr><td align="left" rowspan="1" colspan="1">M7404(cured)</td><td align="left" rowspan="1" colspan="1">DLL3003 (M7404(<italic>tcdC</italic><sup>+</sup>) cured of plasmid pDLL17)</td><td align="left" rowspan="1" colspan="1">This study</td></tr></tbody></table></alternatives></table-wrap></sec><sec id="s4c"><title>Molecular biology and PCR techniques</title><p>Plasmid DNA was isolated using a QIAprep spin miniprep kit (Qiagen). Genomic DNA was prepared using a DNeasy tissue kit (Qiagen). Standard methods for the digestion, modification, ligation, and analysis of plasmid and genomic DNA were used <xref rid="ppat.1002317-Sambrook1" ref-type="bibr">[55]</xref>. Nucleotide sequence analysis was carried out using a PRISM BigDye Terminator cycle sequencing kit (Applied Biosystems) and detection was performed by Micromon at Monash University. Oligonucleotide primer sequences are listed below. Unless otherwise stated, all PCR experiments were carried out with Phusion DNA polymerase (New England Biolabs) and the 2× Failsafe PCR buffer E (Epicentre) according to the manufacturer's instructions.</p></sec><sec id="s4d"><title>Construction of recombinant plasmids</title><p>For construction of the Tn<italic>916</italic> transferrable clostridial shuttle vector, PCR was performed using primers DLP33 (<named-content content-type="gene">5′-GAATTCGCCCTTTTTTATACTCCCCTTG-3′</named-content>) and DLP34 (<named-content content-type="gene">5′-GAATTCGCCCTCAAAGGACGAATATGTCGC-3′</named-content>) and chromosomal DNA extracted from <italic>Clostridium perfringens</italic> strain JIR4225 <xref rid="ppat.1002317-Awad1" ref-type="bibr">[30]</xref>. The resulting 700 bp DNA fragment, which contained the <italic>oriT</italic> region of Tn<italic>916</italic>, was TOPO-cloned into pCR-Blunt II-TOPO according to the manufacturer's instructions (Invitrogen). The fragment was then excised from pCR-Blunt II-TOPO using <italic>Eco</italic>RI and cloned into the equivalent sites of plasmid pMTL9361Cm <xref rid="ppat.1002317-Carter1" ref-type="bibr">[29]</xref>, resulting in plasmid pDLL4.</p><p>For construction of the <italic>tcdC</italic>-carrying plasmid, PCR was performed using primers DLP35 (<named-content content-type="gene">5′-CTGCAGCCACCTCTAAATCACTGAGTCACTTAATTAC-3′</named-content>) and DLP36 (<named-content content-type="gene">5′-CTGCAGAGCCTTGTAACTGTTTATTTGC-3′</named-content>) and <italic>C. difficile</italic> strain VPI10463 genomic DNA in order to amplify a 1085 bp fragment encompassing the <italic>tcdC</italic> gene and upstream region. This fragment was then TOPO-cloned into pCR-Blunt II-TOPO, before being excised with <italic>Pst</italic>I and subcloned into the equivalent site of plasmid pDLL4, resulting in the final construct pDLL17.</p></sec><sec id="s4e"><title>Transfer of plasmid DNA into <italic>C. difficile</italic> by conjugation</title><p>The conjugation procedure utilising <italic>E. coli</italic> HB101(pVS520) as the conjugative donor was carried out as previously described <xref rid="ppat.1002317-Carter1" ref-type="bibr">[29]</xref>. Recombinant plasmids were introduced into <italic>C. perfringens</italic> strain JIR4225 as before <xref rid="ppat.1002317-Awad2" ref-type="bibr">[56]</xref>. Conjugations utilising <italic>C. perfringens</italic> JIR4225 were then performed as follows: separate 90 ml BHIS broth cultures were inoculated with 1 ml aliquots from an overnight <italic>C. difficile</italic> recipient strain or <italic>C. perfringens</italic> donor strain starter culture and grown to mid-exponential phase. Approximately 1 ml was removed from each culture, mixed and centrifuged. The cell pellet was then resuspended in phosphate-buffered saline (PBS), spread onto a BHIS agar plate and incubated overnight at 37°C. Bacterial growth was harvested in sterile PBS before being spread onto BHIS agar supplemented with thiamphenicol and incubated overnight as before. Bacterial growth was again harvested with PBS and dilutions spread onto BHIS agar supplemented with cefoxitin and thiamphenicol or tetracycline, and the plates incubated under anaerobic conditions for 24 to 72 h.</p></sec><sec id="s4f"><title>Toxin A-specific Western blots</title><p>The toxins were partially purified by ammonium sulphate precipitation from culture supernatants harvested after growth for 72 hours and toxin A was then detected by Western blotting as described previously <xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>.</p></sec><sec id="s4g"><title>TcdC-specific Western blots</title><p>For non-quantitative TcdC-specific Western Blots, crude extracts of <italic>C. difficile</italic> were prepared by sonication of samples taken from cultures that had been grown for 12 hours under anaerobic conditions. The crude extracts from each strain were then subjected to electrophoresis in a 15% SDS-PAGE gel and transferred to a nitrocellulose membrane using standard methods <xref rid="ppat.1002317-Sambrook1" ref-type="bibr">[55]</xref>. Membranes were treated with anti-TcdC antibody <xref rid="ppat.1002317-Govind1" ref-type="bibr">[57]</xref> and detected following treatment with goat anti-mouse IgG-alkaline phosphatase conjugated secondary antibody using standard procedures. For quantitative TcdC-specific Western blots, cultures of <italic>C. difficile</italic> VPI10463 and <italic>C. difficile</italic> M7404(<italic>tcdC</italic><sup>+</sup>) were grown under anaerobic conditions and samples were removed every 4 hours for 24 or 28 hours respectively. Each sample was normalized to an optical density (600 nm) of 0.9 prior to lysis, to ensure that the same number of cells was present. Lysates were then prepared by sonication prior to SDS-PAGE gel electrophoresis, transfer and detection, as described above. Following detection, the amount of TcdC in each lysate was quantified by densitometric analysis, using purified recombinant TcdC protein (rTcdC) as the standard and the ImageJ software package, according to published methods <xref rid="ppat.1002317-Abramoff1" ref-type="bibr">[58]</xref>.</p></sec><sec id="s4h"><title>Vero cell cytotoxicity assays</title><p>Toxin B was detected in <italic>C. difficile</italic> culture supernatants harvested after growth for 72 hours by Vero cell cytotoxicity assays as described previously <xref rid="ppat.1002317-Lyras1" ref-type="bibr">[15]</xref>, except that each well was seeded with 1×10<sup>5</sup> cells.</p></sec><sec id="s4i"><title>RNA extraction and reverse transcription</title><p>Total RNA was extracted from <italic>C. difficile</italic> cultures grown for 12 h in TY media. Reverse transcription was performed using AMV Reverse Transcriptase (Promega) using random hexamer oligonucleotides primers and 2 µg template RNA. The cDNA samples were then purified using Qiaquick Columns (Qiagen).</p></sec><sec id="s4j"><title>qRT-PCR assay design and real time PCR</title><p>PaLoc gene specific primers were designed using Primer 3 software (Geneious Software). qRT-PCR was performed using an AB7300 real-time PCR instrument (Applied Biosystems). Reactions were carried out using the FastStart Universal SYBR Green Master Mix (Roche) with 40 ng of cDNA as template. Standard curves were generated for each primer pair using <italic>C. difficile</italic> genomic DNA, and melt curve analysis was performed following each qRT-PCR reaction to verify amplification specificity. Samples were normalised using the <italic>C. difficile rrnA</italic> gene.</p></sec><sec id="s4k"><title>Preparation of spores for animal infection</title><p>Spores were prepared from <italic>C. difficile</italic> cultures grown in 500 ml of BHI broth. Cultures were pelleted by centrifugation for 10 mins and re-suspended in 50% ethanol. The material was then vortexed every 10 min for 1 h before centrifugation for 10 mins. The pellet was then treated with 1% Sarkosyl in PBS for 1 h at room temperature and again pelleted by centrifugation, followed by incubation overnight at 37°C with lysozyme (10 mg/ml) in 125 mM Tris-HCl buffer (pH 8.0). The sample was treated in a sonicating water bath (3 pulses of 3 min each; 1510 Branson) before centrifugation through a 50% sucrose gradient for 20 mins. The pellet was incubated in 2 ml of PBS containing 200 mM EDTA, 300 ng/ml proteinase K and 1% Sarkosyl for 30 mins at 37°C before centrifugation through a 50% sucrose gradient for 20 mins. The final pellet was then washed twice in sterile distilled water before finally being resuspended in 1 ml of sterile water. Spore preparations were stored at −80°C prior to use.</p></sec><sec id="s4l"><title>Hamster experiments</title><p>Female Golden Syrian hamsters purchased from Harlan Olac UK were used for all animal experiments. Telemetry chips (Vitalview Emitter) were inserted by laparotomy into the body cavity of the animals at least 3 weeks before infection with <italic>C. difficile</italic>. Animal experiments were then carried out as described previously <xref rid="ppat.1002317-Goulding1" ref-type="bibr">[33]</xref>, except that animals received 1×10<sup>4</sup> spores of <italic>C. difficile</italic>. Animals were culled when core body temperature dropped below 35°C. This study was carried out in strict accordance with the recommendations in the United Kingdoms Home Office Animals (Scientific Procedures) Act of 1986 which outlines the regulation of the use of laboratory animals for the use of animals in scientific procedures. The experiments described were subject to approval by the University of Glasgow Ethics Committee and by a designated Home Office Inspector (Project Number 60/4218). All experiments were subject to the 3 R consideration (refine, reduce and replace) and all efforts were made to minimize suffering.</p></sec><sec id="s4m"><title>Quantification of bacterial load</title><p>To estimate colonisation, hamsters were sacrificed and the gut region from the caecum to the anus removed. The tissues were homogenised in PBS using a Stomacher and viable counts were performed on the homogenate as described previously <xref rid="ppat.1002317-Goulding1" ref-type="bibr">[33]</xref>.</p></sec><sec id="s4n"><title>Confirmation of infecting strains</title><p>To confirm that the bacteria isolated from the hamster were the same strain as originally used for infection, genomic DNA was isolated and subjected to MVLA as described previously <xref rid="ppat.1002317-Marsh1" ref-type="bibr">[59]</xref>. Plasmid rescue was performed as previously described <xref rid="ppat.1002317-Carter1" ref-type="bibr">[29]</xref> followed by restriction digest analysis to confirm plasmid integrity.</p></sec></sec><sec sec-type="supplementary-material" id="s5"><title>Supporting Information</title><supplementary-material content-type="local-data" id="ppat.1002317.s001"><label>Table S1</label><caption><p><bold>Efficiency of RP4 or Tn</bold><bold><italic>916</italic></bold><bold>-mediated plasmid transfer to </bold><bold><italic>C. difficile</italic></bold><bold> strains.</bold> The efficiency of RP4 or Tn<italic>916</italic>-mediated plasmid transfer from <italic>E. coli</italic> or <italic>C. perfringens</italic> donors, respectively, to <italic>C. difficile</italic> recipient strains is shown, calculated as described in <xref ref-type="sec" rid="s4">Materials and Methods</xref> and expressed as transconjugants per ml.</p><p>(DOC)</p></caption><media xlink:href="ppat.1002317.s001.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="ppat.1002317.s002"><label>Table S2</label><caption><p><bold>Bacterial plasmids.</bold> Bacterial plasmids used in this study, together with the genetic features relevant to this work, are shown.</p><p>(DOC)</p></caption><media xlink:href="ppat.1002317.s002.doc"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><p>We thank Christoph von-Eichel Streiber for kindly providing toxin A-specific antibodies and Rachael Poon and Tongted Phumoonna-Das for assistance with Vero cell cytotoxicity assays.</p></ack><fn-group><fn fn-type="COI-statement"><p>The authors have declared that no competing interests exist.</p></fn><fn fn-type="financial-disclosure"><p>This work was supported by Project Grants from the Australian National Health and Medical Research Council and the Australian Research Council (Monash University), Grant AI057637 from the United States National Institute of Allergy and Infectious Diseases (Monash University and Institut Pasteur) and by Project and Programme Grants from Institut Pasteur, The Wellcome Trust and a personal fellowship for GRD from the Royal Society of Edinburgh (Glasgow University). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p></fn></fn-group><ref-list><title>References</title><ref id="ppat.1002317-Borriello1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Borriello</surname><given-names>SP</given-names></name></person-group><year>1998</year><article-title>Pathogenesis of <italic>Clostridium difficile</italic> infection.</article-title><source>J Antimicrob Chemother</source><volume>41</volume><fpage>13</fpage><lpage>19</lpage></element-citation></ref><ref id="ppat.1002317-Warny1"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Warny</surname><given-names>M</given-names></name><name><surname>Pepin</surname><given-names>J</given-names></name><name><surname>Fang</surname><given-names>A</given-names></name><name><surname>Killgore</surname><given-names>G</given-names></name><name><surname>Thompson</surname><given-names>A</given-names></name><etal></etal></person-group><year>2005</year><article-title>Toxin production by an emerging strain of <italic>Clostridium difficile</italic> associated with outbreaks of severe disease in North America and Europe.</article-title><source>Lancet</source><volume>366</volume><fpage>1079</fpage><lpage>1084</lpage><pub-id pub-id-type="pmid">16182895</pub-id></element-citation></ref><ref id="ppat.1002317-McDonald1"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McDonald</surname><given-names>LC</given-names></name><name><surname>Killgore</surname><given-names>GE</given-names></name><name><surname>Thompson</surname><given-names>A</given-names></name><name><surname>Owens</surname><given-names>RC</given-names></name><name><surname>Kazakova</surname><given-names>SV</given-names></name><etal></etal></person-group><year>2005</year><article-title>An epidemic, toxin gene–variant strain of <italic>Clostridium difficile</italic>.</article-title><source>N Engl J Med</source><volume>353</volume><fpage>2433</fpage><lpage>2441</lpage><pub-id pub-id-type="pmid">16322603</pub-id></element-citation></ref><ref id="ppat.1002317-Stabler1"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stabler</surname><given-names>RA</given-names></name><name><surname>He</surname><given-names>M</given-names></name><name><surname>Dawson</surname><given-names>L</given-names></name><name><surname>Martin</surname><given-names>M</given-names></name><name><surname>Valiente</surname><given-names>E</given-names></name><etal></etal></person-group><year>2009</year><article-title>Comparative genome and phenotypic analysis of <italic>Clostridium difficile</italic> 027 strains provides insight into the evolution of a hypervirulent bacterium.</article-title><source>Genome Biol</source><volume>10</volume><fpage>R102</fpage><pub-id pub-id-type="pmid">19781061</pub-id></element-citation></ref><ref id="ppat.1002317-Loo1"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Loo</surname><given-names>VG</given-names></name><name><surname>Poirier</surname><given-names>L</given-names></name><name><surname>Miller</surname><given-names>MA</given-names></name><name><surname>Oughton</surname><given-names>M</given-names></name><name><surname>Libman</surname><given-names>MD</given-names></name><etal></etal></person-group><year>2005</year><article-title>A predominantly clonal multi-institutional outbreak of <italic>Clostridium difficile</italic>-associated diarrhea with high morbidity and mortality.</article-title><source>N Engl J Med</source><volume>353</volume><fpage>2442</fpage><lpage>2449</lpage><pub-id pub-id-type="pmid">16322602</pub-id></element-citation></ref><ref id="ppat.1002317-Muto1"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Muto</surname><given-names>CA</given-names></name><name><surname>Pokrywka</surname><given-names>M</given-names></name><name><surname>Shutt</surname><given-names>K</given-names></name><name><surname>Mendelsohn</surname><given-names>AB</given-names></name><name><surname>Nouri</surname><given-names>K</given-names></name><etal></etal></person-group><year>2005</year><article-title>A large outbreak of <italic>Clostridium difficile</italic>-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use.</article-title><source>Infect Control Hosp Epidemiol</source><volume>26</volume><fpage>273</fpage><lpage>280</lpage><pub-id pub-id-type="pmid">15796280</pub-id></element-citation></ref><ref id="ppat.1002317-Kuijper1"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuijper</surname><given-names>EJ</given-names></name><name><surname>Coignard</surname><given-names>B</given-names></name><name><surname>Tull</surname><given-names>P</given-names></name></person-group><year>2006</year><article-title>Emergence of <italic>Clostridium difficile</italic>-associated disease in North America and Europe.</article-title><source>Clin Microbiol Infect</source><volume>12</volume><issue>Suppl 6</issue><fpage>2</fpage><lpage>18</lpage></element-citation></ref><ref id="ppat.1002317-Rouphael1"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rouphael</surname><given-names>NG</given-names></name><name><surname>O'Donnell</surname><given-names>JA</given-names></name><name><surname>Bhatnagar</surname><given-names>J</given-names></name><name><surname>Lewis</surname><given-names>F</given-names></name><name><surname>Polgreen</surname><given-names>PM</given-names></name><etal></etal></person-group><year>2008</year><article-title><italic>Clostridium difficile</italic>-associated diarrhea: an emerging threat to pregnant women.</article-title><source>Am J Obstet Gynecol</source><volume>198</volume><fpage>635 e631</fpage><lpage>636</lpage><pub-id pub-id-type="pmid">18395693</pub-id></element-citation></ref><ref id="ppat.1002317-Zilberberg1"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zilberberg</surname><given-names>MD</given-names></name><name><surname>Tillotson</surname><given-names>GS</given-names></name><name><surname>McDonald</surname><given-names>C</given-names></name></person-group><year>2010</year><article-title><italic>Clostridium difficile</italic> infections among hospitalized children, United States, 1997–2006.</article-title><source>Emerg Infect Dis</source><volume>16</volume><fpage>604</fpage><lpage>609</lpage><pub-id pub-id-type="pmid">20350373</pub-id></element-citation></ref><ref id="ppat.1002317-Baker1"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Baker</surname><given-names>SS</given-names></name><name><surname>Faden</surname><given-names>H</given-names></name><name><surname>Sayej</surname><given-names>W</given-names></name><name><surname>Patel</surname><given-names>R</given-names></name><name><surname>Baker</surname><given-names>RD</given-names></name></person-group><year>2010</year><article-title>Increasing Incidence of Community-Associated Atypical <italic>Clostridium difficile</italic> Disease in Children.</article-title><source>Clin Pediatr (Phila)</source><volume>49</volume><fpage>644</fpage><lpage>647</lpage><pub-id pub-id-type="pmid">20150212</pub-id></element-citation></ref><ref id="ppat.1002317-Riley1"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Riley</surname><given-names>TV</given-names></name></person-group><year>2009</year><article-title>Is <italic>Clostridium difficile</italic> a threat to Australia's biosecurity?</article-title><source>Med J Aust</source><volume>190</volume><fpage>661</fpage><lpage>662</lpage><pub-id pub-id-type="pmid">19527197</pub-id></element-citation></ref><ref id="ppat.1002317-Merrigan1"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Merrigan</surname><given-names>M</given-names></name><name><surname>Venugopal</surname><given-names>A</given-names></name><name><surname>Mallozzi</surname><given-names>M</given-names></name><name><surname>Roxas</surname><given-names>B</given-names></name><name><surname>Viswanathan</surname><given-names>VK</given-names></name><etal></etal></person-group><year>2010</year><article-title>Human Hypervirulent <italic>Clostridium difficile</italic> Strains Exhibit Increased Sporulation as Well as Robust Toxin Production.</article-title><source>J Bacteriol</source><volume>192</volume><fpage>4904</fpage><lpage>4911</lpage><pub-id pub-id-type="pmid">20675495</pub-id></element-citation></ref><ref id="ppat.1002317-Sebaihia1"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sebaihia</surname><given-names>M</given-names></name><name><surname>Wren</surname><given-names>BW</given-names></name><name><surname>Mullany</surname><given-names>P</given-names></name><name><surname>Fairweather</surname><given-names>NF</given-names></name><name><surname>Minton</surname><given-names>N</given-names></name><etal></etal></person-group><year>2006</year><article-title>The multidrug-resistant human pathogen <italic>Clostridium difficile</italic> has a highly mobile, mosaic genome.</article-title><source>Nat Genet</source><volume>38</volume><fpage>779</fpage><lpage>786</lpage><pub-id pub-id-type="pmid">16804543</pub-id></element-citation></ref><ref id="ppat.1002317-Just1"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Just</surname><given-names>I</given-names></name><name><surname>Selzer</surname><given-names>J</given-names></name><name><surname>Wilm</surname><given-names>M</given-names></name><name><surname>von Eichel-Streiber</surname><given-names>C</given-names></name><name><surname>Mann</surname><given-names>M</given-names></name><etal></etal></person-group><year>1995</year><article-title>Glucosylation of Rho proteins by <italic>Clostridium difficile</italic> toxin B.</article-title><source>Nature</source><volume>375</volume><fpage>500</fpage><lpage>503</lpage><pub-id pub-id-type="pmid">7777059</pub-id></element-citation></ref><ref id="ppat.1002317-Lyras1"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lyras</surname><given-names>D</given-names></name><name><surname>O'Connor</surname><given-names>JR</given-names></name><name><surname>Howarth</surname><given-names>PK</given-names></name><name><surname>Sambol</surname><given-names>SP</given-names></name><name><surname>Carter</surname><given-names>GP</given-names></name><etal></etal></person-group><year>2009</year><article-title>Toxin B is essential for virulence of <italic>Clostridium difficile</italic>.</article-title><source>Nature</source><volume>458</volume><fpage>1176</fpage><lpage>1179</lpage><pub-id pub-id-type="pmid">19252482</pub-id></element-citation></ref><ref id="ppat.1002317-Kuehne1"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuehne</surname><given-names>SA</given-names></name><name><surname>Cartman</surname><given-names>ST</given-names></name><name><surname>Heap</surname><given-names>JT</given-names></name><name><surname>Kelly</surname><given-names>ML</given-names></name><name><surname>Cockayne</surname><given-names>A</given-names></name><etal></etal></person-group><year>2010</year><article-title>The role of toxin A and toxin B in <italic>Clostridium difficile</italic> infection.</article-title><source>Nature</source><volume>467</volume><fpage>711</fpage><lpage>713</lpage><pub-id pub-id-type="pmid">20844489</pub-id></element-citation></ref><ref id="ppat.1002317-Braun1"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Braun</surname><given-names>V</given-names></name><name><surname>Hundsberger</surname><given-names>T</given-names></name><name><surname>Leukel</surname><given-names>P</given-names></name><name><surname>Sauerborn</surname><given-names>M</given-names></name><name><surname>Eichel-Streiber</surname><given-names>Cv</given-names></name></person-group><year>1996</year><article-title>Definition of the single integration site of the pathogenicity locus in <italic>Clostridium difficile</italic>.</article-title><source>Gene</source><volume>181</volume><fpage>29</fpage><lpage>38</lpage><pub-id pub-id-type="pmid">8973304</pub-id></element-citation></ref><ref id="ppat.1002317-Mani1"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mani</surname><given-names>N</given-names></name><name><surname>Dupuy</surname><given-names>B</given-names></name></person-group><year>2001</year><article-title>Regulation of toxin synthesis in <italic>Clostridium difficile</italic> by an alternative RNA polymerase sigma factor.</article-title><source>Proc Natl Acad Sci U S A</source><volume>98</volume><fpage>5844</fpage><lpage>5849</lpage><pub-id pub-id-type="pmid">11320220</pub-id></element-citation></ref><ref id="ppat.1002317-Tan1"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tan</surname><given-names>KS</given-names></name><name><surname>Wee</surname><given-names>BY</given-names></name><name><surname>Song</surname><given-names>KP</given-names></name></person-group><year>2001</year><article-title>Evidence for holin function of <italic>tcdE</italic> gene in the pathogenicity of <italic>Clostridium difficile</italic>.</article-title><source>J Med Microbiol</source><volume>50</volume><fpage>613</fpage><lpage>619</lpage><pub-id pub-id-type="pmid">11444771</pub-id></element-citation></ref><ref id="ppat.1002317-Matamouros1"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Matamouros</surname><given-names>S</given-names></name><name><surname>England</surname><given-names>P</given-names></name><name><surname>Dupuy</surname><given-names>B</given-names></name></person-group><year>2007</year><article-title><italic>Clostridium difficile</italic> toxin expression is inhibited by the novel regulator TcdC.</article-title><source>Mol Microbiol</source><volume>64</volume><fpage>1274</fpage><lpage>1288</lpage><pub-id pub-id-type="pmid">17542920</pub-id></element-citation></ref><ref id="ppat.1002317-Dupuy1"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dupuy</surname><given-names>B</given-names></name><name><surname>Govind</surname><given-names>R</given-names></name><name><surname>Antunes</surname><given-names>A</given-names></name><name><surname>Matamouros</surname><given-names>S</given-names></name></person-group><year>2008</year><article-title><italic>Clostridium difficile</italic> toxin synthesis is negatively regulated by TcdC.</article-title><source>J Med Microbiol</source><volume>57</volume><fpage>685</fpage><lpage>689</lpage><pub-id pub-id-type="pmid">18480323</pub-id></element-citation></ref><ref id="ppat.1002317-Murray1"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Murray</surname><given-names>R</given-names></name><name><surname>Boyd</surname><given-names>D</given-names></name><name><surname>Levett</surname><given-names>PN</given-names></name><name><surname>Mulvey</surname><given-names>MR</given-names></name><name><surname>Alfa</surname><given-names>MJ</given-names></name></person-group><year>2009</year><article-title>Truncation in the <italic>tcdC</italic> region of the <italic>Clostridium difficile</italic> PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A.</article-title><source>BMC Infect Dis</source><volume>9</volume><fpage>103</fpage><pub-id pub-id-type="pmid">19558711</pub-id></element-citation></ref><ref id="ppat.1002317-Mani2"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mani</surname><given-names>N</given-names></name><name><surname>Lyras</surname><given-names>D</given-names></name><name><surname>Barroso</surname><given-names>L</given-names></name><name><surname>Howarth</surname><given-names>P</given-names></name><name><surname>Wilkins</surname><given-names>T</given-names></name><etal></etal></person-group><year>2002</year><article-title>Environmental response and autoregulation of <italic>Clostridium difficile</italic> TxeR, a sigma factor for toxin gene expression.</article-title><source>J Bacteriol</source><volume>184</volume><fpage>5971</fpage><lpage>5978</lpage><pub-id pub-id-type="pmid">12374831</pub-id></element-citation></ref><ref id="ppat.1002317-OConnor1"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>O'Connor</surname><given-names>JR</given-names></name><name><surname>Lyras</surname><given-names>D</given-names></name><name><surname>Farrow</surname><given-names>KA</given-names></name><name><surname>Adams</surname><given-names>V</given-names></name><name><surname>Powell</surname><given-names>DR</given-names></name><etal></etal></person-group><year>2006</year><article-title>Construction and analysis of chromosomal <italic>Clostridium difficile</italic> mutants.</article-title><source>Mol Microbiol</source><volume>61</volume><fpage>1335</fpage><lpage>1351</lpage><pub-id pub-id-type="pmid">16925561</pub-id></element-citation></ref><ref id="ppat.1002317-Burns1"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Burns</surname><given-names>DA</given-names></name><name><surname>Heap</surname><given-names>JT</given-names></name><name><surname>Minton</surname><given-names>NP</given-names></name></person-group><year>2010</year><article-title>SleC is essential for germination of <italic>Clostridium difficile</italic> spores in nutrient-rich medium supplemented with the bile salt taurocholate.</article-title><source>J Bacteriol</source><volume>192</volume><fpage>657</fpage><lpage>664</lpage><pub-id pub-id-type="pmid">19933358</pub-id></element-citation></ref><ref id="ppat.1002317-Cartman1"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cartman</surname><given-names>ST</given-names></name><name><surname>Minton</surname><given-names>NP</given-names></name></person-group><year>2010</year><article-title>A mariner-based transposon system for <italic>in vivo</italic> random mutagenesis of <italic>Clostridium difficile</italic>.</article-title><source>Appl Environ Microbiol</source><volume>76</volume><fpage>1103</fpage><lpage>1109</lpage><pub-id pub-id-type="pmid">20023081</pub-id></element-citation></ref><ref id="ppat.1002317-Heap1"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heap</surname><given-names>JT</given-names></name><name><surname>Kuehne</surname><given-names>SA</given-names></name><name><surname>Ehsaan</surname><given-names>M</given-names></name><name><surname>Cartman</surname><given-names>ST</given-names></name><name><surname>Cooksley</surname><given-names>CM</given-names></name><etal></etal></person-group><year>2010</year><article-title>The ClosTron: Mutagenesis in <italic>Clostridium</italic> refined and streamlined.</article-title><source>J Microbiol Methods</source><volume>80</volume><fpage>49</fpage><lpage>55</lpage><pub-id pub-id-type="pmid">19891996</pub-id></element-citation></ref><ref id="ppat.1002317-Nallapareddy1"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nallapareddy</surname><given-names>SR</given-names></name><name><surname>Singh</surname><given-names>KV</given-names></name><name><surname>Murray</surname><given-names>BE</given-names></name></person-group><year>2006</year><article-title>Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical <italic>Enterococcus faecium</italic> strains.</article-title><source>Appl Environ Microbiol</source><volume>72</volume><fpage>334</fpage><lpage>345</lpage><pub-id pub-id-type="pmid">16391062</pub-id></element-citation></ref><ref id="ppat.1002317-Carter1"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carter</surname><given-names>GP</given-names></name><name><surname>Lyras</surname><given-names>D</given-names></name><name><surname>Allen</surname><given-names>DL</given-names></name><name><surname>Mackin</surname><given-names>KE</given-names></name><name><surname>Howarth</surname><given-names>PM</given-names></name><etal></etal></person-group><year>2007</year><article-title>Binary toxin production in <italic>Clostridium difficile</italic> is regulated by CdtR, a LytTR family response regulator.</article-title><source>J Bacteriol</source><volume>189</volume><fpage>7290</fpage><lpage>7301</lpage><pub-id pub-id-type="pmid">17693517</pub-id></element-citation></ref><ref id="ppat.1002317-Awad1"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Awad</surname><given-names>MM</given-names></name><name><surname>Rood</surname><given-names>JI</given-names></name></person-group><year>1997</year><article-title>Isolation of a-toxin, q-toxin and k-toxin mutants of <italic>Clostridium perfringens</italic> by Tn<italic>916</italic> mutagenesis.</article-title><source>Microb Pathogen</source><volume>22</volume><fpage>275</fpage><lpage>284</lpage><pub-id pub-id-type="pmid">9160297</pub-id></element-citation></ref><ref id="ppat.1002317-Hundsberger1"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hundsberger</surname><given-names>T</given-names></name><name><surname>Braun</surname><given-names>V</given-names></name><name><surname>Weidmann</surname><given-names>M</given-names></name><name><surname>Leukel</surname><given-names>P</given-names></name><name><surname>Sauerborn</surname><given-names>M</given-names></name><etal></etal></person-group><year>1997</year><article-title>Transcription analysis of the genes <italic>tcdA-E</italic> of the pathogenicity locus of <italic>Clostridium difficile</italic>.</article-title><source>European J Bioch</source><volume>244</volume><fpage>735</fpage><lpage>742</lpage></element-citation></ref><ref id="ppat.1002317-Razaq1"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Razaq</surname><given-names>N</given-names></name><name><surname>Sambol</surname><given-names>S</given-names></name><name><surname>Nagaro</surname><given-names>K</given-names></name><name><surname>Zukowski</surname><given-names>W</given-names></name><name><surname>Cheknis</surname><given-names>A</given-names></name><etal></etal></person-group><year>2007</year><article-title>Infection of hamsters with historical and epidemic BI types of <italic>Clostridium difficile</italic>.</article-title><source>J Infect Dis</source><volume>196</volume><fpage>1813</fpage><lpage>1819</lpage><pub-id pub-id-type="pmid">18190262</pub-id></element-citation></ref><ref id="ppat.1002317-Goulding1"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goulding</surname><given-names>D</given-names></name><name><surname>Thompson</surname><given-names>H</given-names></name><name><surname>Emerson</surname><given-names>J</given-names></name><name><surname>Fairweather</surname><given-names>NF</given-names></name><name><surname>Dougan</surname><given-names>G</given-names></name><etal></etal></person-group><year>2009</year><article-title>Distinctive profiles of infection and pathology in hamsters infected with <italic>Clostridium difficile</italic> strains 630 and B1.</article-title><source>Infect Immun</source><volume>77</volume><fpage>5478</fpage><lpage>5485</lpage><pub-id pub-id-type="pmid">19752031</pub-id></element-citation></ref><ref id="ppat.1002317-He1"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>He</surname><given-names>M</given-names></name><name><surname>Sebaihia</surname><given-names>M</given-names></name><name><surname>Lawley</surname><given-names>TD</given-names></name><name><surname>Stabler</surname><given-names>RA</given-names></name><name><surname>Dawson</surname><given-names>LF</given-names></name><etal></etal></person-group><year>2010</year><article-title>Evolutionary dynamics of <italic>Clostridium difficile</italic> over short and long time scales.</article-title><source>Proc Natl Acad Sci U S A</source><volume>107</volume><fpage>7527</fpage><lpage>7532</lpage><pub-id pub-id-type="pmid">20368420</pub-id></element-citation></ref><ref id="ppat.1002317-Rupnik1"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rupnik</surname><given-names>M</given-names></name><name><surname>Avesani</surname><given-names>V</given-names></name><name><surname>Janc</surname><given-names>M</given-names></name><name><surname>von Eichel-Streiber</surname><given-names>C</given-names></name><name><surname>Delmee</surname><given-names>M</given-names></name></person-group><year>1998</year><article-title>A novel toxinotyping scheme and correlation of toxinotypes with serogroups of <italic>Clostridium difficile</italic> isolates.</article-title><source>J Clin Microbiol</source><volume>36</volume><fpage>2240</fpage><lpage>2247</lpage><pub-id pub-id-type="pmid">9665999</pub-id></element-citation></ref><ref id="ppat.1002317-Richards1"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Richards</surname><given-names>M</given-names></name><name><surname>Knox</surname><given-names>J</given-names></name><name><surname>Elliott</surname><given-names>B</given-names></name><name><surname>Mackin</surname><given-names>K</given-names></name><name><surname>Lyras</surname><given-names>D</given-names></name><etal></etal></person-group><year>2011</year><article-title>Severe infection with Clostridium difficile PCR ribotype 027 acquired in Melbourne, Australia.</article-title><source>Med J Australia</source><volume>194</volume><fpage>369</fpage><lpage>371</lpage><pub-id pub-id-type="pmid">21470090</pub-id></element-citation></ref><ref id="ppat.1002317-Curry1"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Curry</surname><given-names>SR</given-names></name><name><surname>Marsh</surname><given-names>JW</given-names></name><name><surname>Muto</surname><given-names>CA</given-names></name><name><surname>O'Leary</surname><given-names>MM</given-names></name><name><surname>Pasculle</surname><given-names>AW</given-names></name><etal></etal></person-group><year>2007</year><article-title><italic>tcdC</italic> genotypes associated with severe TcdC truncation in an epidemic clone and other strains of <italic>Clostridium difficile</italic>.</article-title><source>J Clin Microbiol</source><volume>45</volume><fpage>215</fpage><lpage>221</lpage><pub-id pub-id-type="pmid">17035492</pub-id></element-citation></ref><ref id="ppat.1002317-Lyerly1"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lyerly</surname><given-names>DM</given-names></name><name><surname>Barroso</surname><given-names>LA</given-names></name><name><surname>Wilkins</surname><given-names>TD</given-names></name><name><surname>Depitre</surname><given-names>C</given-names></name><name><surname>Corthier</surname><given-names>G</given-names></name></person-group><year>1992</year><article-title>Characterization of a toxin A-negative, toxin B-positive strain of <italic>Clostridium difficile</italic>.</article-title><source>Infect Immun</source><volume>60</volume><fpage>4633</fpage><lpage>4639</lpage><pub-id pub-id-type="pmid">1398977</pub-id></element-citation></ref><ref id="ppat.1002317-Carter2"><label>39</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carter</surname><given-names>GP</given-names></name><name><surname>Rood</surname><given-names>JI</given-names></name><name><surname>Lyras</surname><given-names>D</given-names></name></person-group><year>2010</year><article-title>The role of toxin A and toxin B in <italic>Clostridium difficile</italic>-associated disease: Past and present perspectives.</article-title><source>Gut Microbes</source><volume>1</volume><fpage>58</fpage><lpage>64</lpage><pub-id pub-id-type="pmid">20664812</pub-id></element-citation></ref><ref id="ppat.1002317-OConnor2"><label>40</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>O'Connor</surname><given-names>JR</given-names></name><name><surname>Johnson</surname><given-names>S</given-names></name><name><surname>Gerding</surname><given-names>DN</given-names></name></person-group><year>2009</year><article-title><italic>Clostridium difficile</italic> infection caused by the epidemic BI/NAP1/027 strain.</article-title><source>Gastroenterology</source><volume>136</volume><fpage>1913</fpage><lpage>1924</lpage><pub-id pub-id-type="pmid">19457419</pub-id></element-citation></ref><ref id="ppat.1002317-Heap2"><label>41</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heap</surname><given-names>JT</given-names></name><name><surname>Pennington</surname><given-names>OJ</given-names></name><name><surname>Cartman</surname><given-names>ST</given-names></name><name><surname>Carter</surname><given-names>GP</given-names></name><name><surname>NP.</surname><given-names>M</given-names></name></person-group><year>2007</year><article-title>The ClosTron: a universal gene knock-out system for the genus <italic>Clostridium</italic>.</article-title><source>J Microbiol Meth</source><volume>70</volume><fpage>452</fpage><lpage>464</lpage></element-citation></ref><ref id="ppat.1002317-Mullany1"><label>42</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mullany</surname><given-names>P</given-names></name><name><surname>Wilks</surname><given-names>M</given-names></name><name><surname>Tabaqchali</surname><given-names>S</given-names></name></person-group><year>1991</year><article-title>Transfer of Tn<italic>916</italic> and Tn<italic>916</italic>DE into <italic>Clostridium difficile</italic>: demonstration of a hot-spot for these elemnets in the <italic>C. difficile</italic> genome.</article-title><source>FEMS Microbiol Lett</source><volume>79</volume><fpage>191</fpage><lpage>194</lpage></element-citation></ref><ref id="ppat.1002317-Minton1"><label>43</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Minton</surname><given-names>N</given-names></name><name><surname>Carter</surname><given-names>G</given-names></name><name><surname>Herbert</surname><given-names>M</given-names></name><name><surname>O'Keeffe</surname><given-names>T</given-names></name><name><surname>Purdy</surname><given-names>D</given-names></name><etal></etal></person-group><year>2004</year><article-title>The development of <italic>Clostridium difficile</italic> genetic systems.</article-title><source>Anaerobe</source><volume>10</volume><fpage>75</fpage><lpage>84</lpage><pub-id pub-id-type="pmid">16701503</pub-id></element-citation></ref><ref id="ppat.1002317-Goorhuis1"><label>44</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Goorhuis</surname><given-names>A</given-names></name><name><surname>Debast</surname><given-names>SB</given-names></name><name><surname>van Leengoed</surname><given-names>LA</given-names></name><name><surname>Harmanus</surname><given-names>C</given-names></name><name><surname>Notermans</surname><given-names>DW</given-names></name><etal></etal></person-group><year>2008</year><article-title><italic>Clostridium difficile</italic> PCR ribotype 078: an emerging strain in humans and in pigs?</article-title><source>J Clin Microbiol</source><volume>46</volume><fpage>1157; author reply 1158</fpage><pub-id pub-id-type="pmid">18326836</pub-id></element-citation></ref><ref id="ppat.1002317-Dawson1"><label>45</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dawson</surname><given-names>LF</given-names></name><name><surname>Valiente</surname><given-names>E</given-names></name><name><surname>Wren</surname><given-names>BW</given-names></name></person-group><year>2009</year><article-title><italic>Clostridium difficile</italic>–a continually evolving and problematic pathogen.</article-title><source>Infect Genet Evol</source><volume>9</volume><fpage>1410</fpage><lpage>1417</lpage><pub-id pub-id-type="pmid">19539054</pub-id></element-citation></ref><ref id="ppat.1002317-Stabler2"><label>46</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stabler</surname><given-names>RA</given-names></name><name><surname>Dawson</surname><given-names>LF</given-names></name><name><surname>Phua</surname><given-names>LT</given-names></name><name><surname>Wren</surname><given-names>BW</given-names></name></person-group><year>2008</year><article-title>Comparative analysis of BI/NAP1/027 hypervirulent strains reveals novel toxin B-encoding gene (<italic>tcdB</italic>) sequences.</article-title><source>J Med Microbiol</source><volume>57</volume><fpage>771</fpage><lpage>775</lpage><pub-id pub-id-type="pmid">18480336</pub-id></element-citation></ref><ref id="ppat.1002317-Lanis1"><label>47</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lanis</surname><given-names>JM</given-names></name><name><surname>Barua</surname><given-names>S</given-names></name><name><surname>Ballard</surname><given-names>JD</given-names></name></person-group><year>2010</year><article-title>Variations in TcdB activity and the hypervirulence of emerging strains of <italic>Clostridium difficile</italic>.</article-title><source>PLoS Pathog</source><volume>6</volume><fpage>e1001061</fpage><pub-id pub-id-type="pmid">20808849</pub-id></element-citation></ref><ref id="ppat.1002317-Schwan1"><label>48</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schwan</surname><given-names>C</given-names></name><name><surname>Stecher</surname><given-names>B</given-names></name><name><surname>Tzivelekidis</surname><given-names>T</given-names></name><name><surname>van Ham</surname><given-names>M</given-names></name><name><surname>Rohde</surname><given-names>M</given-names></name><etal></etal></person-group><year>2009</year><article-title><italic>Clostridium difficile</italic> toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria.</article-title><source>PLoS Pathog</source><volume>5</volume><fpage>e1000626</fpage><pub-id pub-id-type="pmid">19834554</pub-id></element-citation></ref><ref id="ppat.1002317-Akerlund1"><label>49</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Akerlund</surname><given-names>T</given-names></name><name><surname>Persson</surname><given-names>I</given-names></name><name><surname>Unemo</surname><given-names>M</given-names></name><name><surname>Noren</surname><given-names>T</given-names></name><name><surname>Svenungsson</surname><given-names>B</given-names></name><etal></etal></person-group><year>2008</year><article-title>Increased sporulation rate of epidemic <italic>Clostridium difficile</italic> Type 027/NAP1.</article-title><source>J Clin Microbiol</source><volume>46</volume><fpage>1530</fpage><lpage>1533</lpage><pub-id pub-id-type="pmid">18287318</pub-id></element-citation></ref><ref id="ppat.1002317-Lawley1"><label>50</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lawley</surname><given-names>TD</given-names></name><name><surname>Clare</surname><given-names>S</given-names></name><name><surname>Walker</surname><given-names>AW</given-names></name><name><surname>Goulding</surname><given-names>D</given-names></name><name><surname>Stabler</surname><given-names>RA</given-names></name><etal></etal></person-group><year>2009</year><article-title>Antibiotic treatment of <italic>Clostridium difficile</italic> carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts.</article-title><source>Infect Immun</source><volume>77</volume><fpage>3661</fpage><lpage>3669</lpage><pub-id pub-id-type="pmid">19564382</pub-id></element-citation></ref><ref id="ppat.1002317-Johnson1"><label>51</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Johnson</surname><given-names>S</given-names></name></person-group><year>2009</year><article-title>Recurrent <italic>Clostridium difficile</italic> infection: a review of risk factors, treatments, and outcomes.</article-title><source>J Infect</source><volume>58</volume><fpage>403</fpage><lpage>410</lpage><pub-id pub-id-type="pmid">19394704</pub-id></element-citation></ref><ref id="ppat.1002317-Lindsay1"><label>52</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lindsay</surname><given-names>JA</given-names></name></person-group><year>2010</year><article-title>Genomic variation and evolution of <italic>Staphylococcus aureus</italic>.</article-title><source>Int J Med Microbiol</source><volume>300</volume><fpage>98</fpage><lpage>103</lpage><pub-id pub-id-type="pmid">19811948</pub-id></element-citation></ref><ref id="ppat.1002317-Bonten1"><label>53</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bonten</surname><given-names>MJ</given-names></name><name><surname>Willems</surname><given-names>R</given-names></name><name><surname>Weinstein</surname><given-names>RA</given-names></name></person-group><year>2001</year><article-title>Vancomycin-resistant enterococci: why are they here, and where do they come from?</article-title><source>Lancet Infect Dis</source><volume>1</volume><fpage>314</fpage><lpage>325</lpage><pub-id pub-id-type="pmid">11871804</pub-id></element-citation></ref><ref id="ppat.1002317-Smith1"><label>54</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Smith</surname><given-names>CJ</given-names></name><name><surname>Markowitz</surname><given-names>SM</given-names></name><name><surname>Macrina</surname><given-names>FL</given-names></name></person-group><year>1981</year><article-title>Transferable tetracycline resistance in <italic>Clostridium difficile</italic>.</article-title><source>Antimicrob Agents and Chemother</source><volume>19</volume><fpage>997</fpage><lpage>1003</lpage><pub-id pub-id-type="pmid">7271279</pub-id></element-citation></ref><ref id="ppat.1002317-Sambrook1"><label>55</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Sambrook</surname><given-names>J</given-names></name><name><surname>Russell</surname><given-names>DW</given-names></name></person-group><year>2001</year><source>Molecular Cloning: A Laboratory Manual</source><publisher-loc>Cold Spring Harbor, NY</publisher-loc><publisher-name>Cold Spring Harbor Laboratory Press</publisher-name></element-citation></ref><ref id="ppat.1002317-Awad2"><label>56</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Awad</surname><given-names>MM</given-names></name><name><surname>Bryant</surname><given-names>AE</given-names></name><name><surname>Stevens</surname><given-names>DL</given-names></name><name><surname>Rood</surname><given-names>JI</given-names></name></person-group><year>1995</year><article-title>Virulence studies on chromosomal a-toxin and q-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of a-toxin in <italic>Clostridium perfringens</italic>-mediated gas gangrene.</article-title><source>Mol Microbiol</source><volume>15</volume><fpage>191</fpage><lpage>202</lpage><pub-id pub-id-type="pmid">7746141</pub-id></element-citation></ref><ref id="ppat.1002317-Govind1"><label>57</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Govind</surname><given-names>R</given-names></name><name><surname>Vediyappan</surname><given-names>G</given-names></name><name><surname>Rolfe</surname><given-names>RD</given-names></name><name><surname>Fralick</surname><given-names>JA</given-names></name></person-group><year>2006</year><article-title>Evidence that <italic>Clostridium difficile</italic> TcdC is a membrane-associated protein.</article-title><source>J Bacteriol</source><volume>188</volume><fpage>3716</fpage><lpage>3720</lpage><pub-id pub-id-type="pmid">16672625</pub-id></element-citation></ref><ref id="ppat.1002317-Abramoff1"><label>58</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Abramoff</surname><given-names>MD</given-names></name><name><surname>Magelhaes</surname><given-names>PJ</given-names></name><name><surname>Ram</surname><given-names>SJ</given-names></name></person-group><year>2004</year><article-title>Image Processing with ImageJ.</article-title><source>Biophotonics Internatl</source><volume>11</volume><fpage>36</fpage><lpage>42</lpage></element-citation></ref><ref id="ppat.1002317-Marsh1"><label>59</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Marsh</surname><given-names>JW</given-names></name><name><surname>O'Leary</surname><given-names>MM</given-names></name><name><surname>Shutt</surname><given-names>KA</given-names></name><name><surname>Pasculle</surname><given-names>AW</given-names></name><name><surname>Johnson</surname><given-names>S</given-names></name><etal></etal></person-group><year>2006</year><article-title>Multilocus variable-number tandem-repeat analysis for investigation of <italic>Clostridium difficile</italic> transmission in Hospitals.</article-title><source>J Clin Microbiol</source><volume>44</volume><fpage>2558</fpage><lpage>2566</lpage><pub-id pub-id-type="pmid">16825380</pub-id></element-citation></ref><ref id="ppat.1002317-Boyer1"><label>60</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Boyer</surname><given-names>HW</given-names></name><name><surname>Roulland-Dussoix</surname><given-names>D</given-names></name></person-group><year>1969</year><article-title>A complementation analysis of the restriction and modification of DNA in <italic>Escherichia coli</italic>.</article-title><source>J Mol Biol</source><volume>41</volume><fpage>459</fpage><lpage>472</lpage><pub-id pub-id-type="pmid">4896022</pub-id></element-citation></ref><ref id="ppat.1002317-Wust1"><label>61</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wust</surname><given-names>J</given-names></name><name><surname>Sullivan</surname><given-names>NM</given-names></name><name><surname>Hardegger</surname><given-names>U</given-names></name><name><surname>Wilkins</surname><given-names>TD</given-names></name></person-group><year>1982</year><article-title>Investigation of an outbreak of antibiotic-associated colitis by various typing methods.</article-title><source>J Clin Microbiol</source><volume>16</volume><fpage>1096</fpage><lpage>1101</lpage><pub-id pub-id-type="pmid">7161375</pub-id></element-citation></ref><ref id="ppat.1002317-Mullany2"><label>62</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mullany</surname><given-names>P</given-names></name><name><surname>Wilks</surname><given-names>M</given-names></name><name><surname>Lamb</surname><given-names>I</given-names></name><name><surname>Clayton</surname><given-names>C</given-names></name><name><surname>Wren</surname><given-names>B</given-names></name><etal></etal></person-group><year>1990</year><article-title>Genetic analysis of a tetracycline resistance element from <italic>Clostridium difficile</italic> and its conjugal transfer to and from <italic>Bacillus subtilis</italic>.</article-title><source>J Gen Microbiol</source><volume>136</volume><fpage>1343</fpage><lpage>1349</lpage><pub-id pub-id-type="pmid">2172445</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Wicri/Asie/explor/AustralieFrV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 002C57 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 002C57 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Wicri/Asie
|area= AustralieFrV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.33. |  |