1887

Abstract

Rapid identification of hypervirulent strains is essential for preventing their spread. Recent completion of several full-length genomes provided an excellent opportunity to identify potentially unique genes that characterize hypervirulent strains. Based on sequence comparisons between strains we describe two gene insertions into the genome of hypervirulent PCR ribotypes 078 and 027. Analysis of these regions, of 1.7 and 4.2 kb, respectively, revealed that they contain several interesting ORFs. The 078 region is inserted intergenically and introduces an enzyme that is involved in the biosynthesis of several antibiotics. The 027 insert disrupts the thymidylate synthetase () gene and replaces it with an equivalent, catalytically more efficient, gene. Both gene insertions were used to develop ribotype-specific PCRs, which were validated by screening a large strain collection consisting of 68 different PCR ribotypes supplemented with diverse 078 and 027 strains derived from different geographical locations and individual outbreaks. The genetic markers were stably present in the hypervirulent PCR ribotypes 078 and 027, but were also found in several other PCR ribotypes. Comparative analysis of amplified fragment length polymorphisms, PCR ribotype banding patterns and toxin profiles showed that all PCR ribotypes sharing the same insert from phylogenetically coherent clusters. The identified loci are unique to these clusters, to which the hypervirulent ribotypes 078 and 027 belong. This provides valuable information on strains belonging to two distinct lineages within that are highly related to hypervirulent strains.

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2011-11-01
2020-04-07
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References

  1. Akerlund T., Persson I., Unemo M., Norén T., Svenungsson B., Wullt M., Burman L. G..( 2008;). Increased sporulation rate of epidemic Clostridium difficile type 027/NAP1. J Clin Microbiol46:1530–1533 [CrossRef][PubMed]
    [Google Scholar]
  2. Alonso R., Muñoz C., Gros S., García de Viedma D., Peláez T., Bouza E..( 1999;). Rapid detection of toxigenic Clostridium difficile from stool samples by a nested PCR of toxin B gene. J Hosp Infect41:145–149 [CrossRef][PubMed]
    [Google Scholar]
  3. Avbersek J., Janezic S., Pate M., Rupnik M., Zidaric V., Logar K., Vengust M., Zemljic M., Pirs T., Ocepek M..( 2009;). Diversity of Clostridium difficile in pigs and other animals in Slovenia. Anaerobe15:252–255 [CrossRef][PubMed]
    [Google Scholar]
  4. Bakker D., Corver J., Harmanus C., Goorhuis A., Keessen E. C., Fawley W. N., Wilcox M. H., Kuijper E. J..( 2010;). Relatedness of human and animal Clostridium difficile PCR ribotype 078 isolates determined on the basis of multilocus variable-number tandem-repeat analysis and tetracycline resistance. J Clin Microbiol48:3744–3749 [CrossRef][PubMed]
    [Google Scholar]
  5. Bauer M. P., Notermans D. W., van Benthem B. H., Brazier J. S., Wilcox M. H., Rupnik M., Monnet D. L., van Dissel J. T., Kuijper E. J..ECDIS Study Group( 2011;). Clostridium difficile infection in Europe: a hospital-based survey. Lancet377:63–73 [CrossRef][PubMed]
    [Google Scholar]
  6. Bidet P., Barbut F., Lalande V., Burghoffer B., Petit J. C..( 1999;). Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett175:261–266 [CrossRef][PubMed]
    [Google Scholar]
  7. Brazier J. S..( 2001;). Typing of Clostridium difficile. Clin Microbiol Infect7:428–431 [CrossRef][PubMed]
    [Google Scholar]
  8. Chaudhuri R. R., Loman N. J., Snyder L. A., Bailey C. M., Stekel D. J., Pallen M. J..( 2008;). xBASE2: a comprehensive resource for comparative bacterial genomics. Nucleic Acids Res36:Database issueD543–D546 [CrossRef][PubMed]
    [Google Scholar]
  9. Dingle K. E., Griffiths D., Didelot X., Evans J., Vaughan A., Kachrimanidou M., Stoesser N., Jolley K. A., Golubchik T. et al.& other authors ( 2011;). Clinical Clostridium difficile: clonality and pathogenicity locus diversity. PLoS ONE6:e19993 [CrossRef][PubMed]
    [Google Scholar]
  10. Escartin F., Skouloubris S., Liebl U., Myllykallio H..( 2008;). Flavin-dependent thymidylate synthase X limits chromosomal DNA replication. Proc Natl Acad Sci U S A105:9948–9952 [CrossRef][PubMed]
    [Google Scholar]
  11. Esra Önen F., Boum Y., Jacquement C., Spanedda M. V., Jaber N., Scherman D., Myllykallio H., Herscovici J..( 2008;). Design, synthesis and evaluation of potent thymidylate synthase X inhibitors. Bioorg Med Chem Lett18:3628–3631 [CrossRef][PubMed]
    [Google Scholar]
  12. Goorhuis A., Bakker D., Corver J., Debast S. B., Harmanus C., Notermans D. W., Bergwerff A. A., Dekker F. W., Kuijper E. J..( 2008;). Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis47:1162–1170 [CrossRef][PubMed]
    [Google Scholar]
  13. Griffiths D., Fawley W., Kachrimanidou M., Bowden R., Crook D. W., Fung R., Golubchik T., Harding R. M., Jeffery K. J. et al.& other authors ( 2010;). Multilocus sequence typing of Clostridium difficile. J Clin Microbiol48:770–778 [CrossRef][PubMed]
    [Google Scholar]
  14. He M., Sebaihia M., Lawley T. D., Stabler R. A., Dawson L. F., Martin M. J., Holt K. E., Seth-Smith H. M., Quail M. A. et al.& other authors ( 2010;). Evolutionary dynamics of Clostridium difficile over short and long time scales. Proc Natl Acad Sci U S A107:7527–7532 [CrossRef][PubMed]
    [Google Scholar]
  15. Indra A., Blaschitz M., Kernbichler S., Reischl U., Wewalka G., Allerberger F..( 2010;). Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region. J Med Microbiol59:1317–1323 [CrossRef][PubMed]
    [Google Scholar]
  16. Janezic S., Strumbelj I., Rupnik M..( 2011;). Use of modified PCR ribotyping for direct detection of Clostridium difficile ribotypes in stool samples. J Clin Microbiol49:3024–3025 [CrossRef][PubMed]
    [Google Scholar]
  17. Janvilisri T., Scaria J., Thompson A. D., Nicholson A., Limbago B. M., Arroyo L. G., Songer J. G., Gröhn Y. T., Chang Y. F..( 2009;). Microarray identification of Clostridium difficile core components and divergent regions associated with host origin. J Bacteriol191:3881–3891 [CrossRef][PubMed]
    [Google Scholar]
  18. Kallen A. J., Thompson A., Ristaino P., Chapman L., Nicholson A., Sim B. T., Lessa F., Sharapov U., Fadden E. et al.& other authors ( 2009;). Complete restriction of fluoroquinolone use to control an outbreak of Clostridium difficile infection at a community hospital. Infect Control Hosp Epidemiol30:264–272 [CrossRef][PubMed]
    [Google Scholar]
  19. Kato H., Kato N., Katow S., Maegawa T., Nakamura S., Lyerly D. M..( 1999;). Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains. FEMS Microbiol Lett175:197–203 [CrossRef][PubMed]
    [Google Scholar]
  20. Keel K., Brazier J. S., Post K. W., Weese S., Songer J. G..( 2007;). Prevalence of PCR ribotypes among Clostridium difficile isolates from pigs, calves, and other species. J Clin Microbiol45:1963–1964 [CrossRef][PubMed]
    [Google Scholar]
  21. Kelly C. P., Pothoulakis C., LaMont J. T..( 1994;). Clostridium difficile colitis. N Engl J Med330:257–262 [CrossRef][PubMed]
    [Google Scholar]
  22. Killgore G., Thompson A., Johnson S., Brazier J., Kuijper E., Pepin J., Frost E. H., Savelkoul P., Nicholson B. et al.& other authors ( 2008;). Comparison of seven techniques for typing international epidemic strains of Clostridium difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing. J Clin Microbiol46:431–437 [CrossRef][PubMed]
    [Google Scholar]
  23. Koehn E. M., Kohen A..( 2010;). Flavin-dependent thymidylate synthase: a novel pathway towards thymine. Arch Biochem Biophys493:96–102 [CrossRef][PubMed]
    [Google Scholar]
  24. Koehn E. M., Fleischmann T., Conrad J. A., Palfey B. A., Lesley S. A., Mathews I. I., Kohen A..( 2009;). An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene. Nature458:919–923 [CrossRef][PubMed]
    [Google Scholar]
  25. Lyras D., O’Connor J. R., Howarth P. M., Sambol S. P., Carter G. P., Phumoonna T., Poon R., Adams V., Vedantam G. et al.& other authors ( 2009;). Toxin B is essential for virulence of Clostridium difficile. Nature458:1176–1179 [CrossRef][PubMed]
    [Google Scholar]
  26. Marsden G. L., Davis I. J., Wright V. J., Sebaihia M., Kuijper E. J., Minton N. P..( 2010;). Array comparative hybridisation reveals a high degree of similarity between UK and European clinical isolates of hypervirulent Clostridium difficile. BMC Genomics11:389 [CrossRef][PubMed]
    [Google Scholar]
  27. McDonald L. C., Killgore G. E., Thompson A., Owens R. C. Jr, Kazakova S. V., Sambol S. P., Johnson S., Gerding D. N..( 2005;). An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med353:2433–2441 [CrossRef][PubMed]
    [Google Scholar]
  28. Merrigan M., Venugopal A., Mallozzi M., Roxas B., Viswanathan V. K., Johnson S., Gerding D. N., Vedantam G..( 2010;). Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production. J Bacteriol192:4904–4911 [CrossRef][PubMed]
    [Google Scholar]
  29. Pépin J., Valiquette L., Alary M. E., Villemure P., Pelletier A., Forget K., Pépin K., Chouinard D..( 2004;). Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ171:466–472 [CrossRef][PubMed]
    [Google Scholar]
  30. Pépin J., Valiquette L., Cossette B..( 2005;). Mortality attributable to nosocomial Clostridium difficile-associated disease during an epidemic caused by a hypervirulent strain in Quebec. CMAJ173:1037–1042 [CrossRef][PubMed]
    [Google Scholar]
  31. Pirs T., Ocepek M., Rupnik M..( 2008;). Isolation of Clostridium difficile from food animals in Slovenia. J Med Microbiol57:790–792 [CrossRef][PubMed]
    [Google Scholar]
  32. Pituch H., Brazier J. S., Obuch-Woszczatynski P., Wultanska D., Meisel-Mikolajczyk F., Luczak M..( 2006;). Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance. J Med Microbiol55:207–213 [CrossRef][PubMed]
    [Google Scholar]
  33. Rupnik M., Brazier J. S., Duerden B. I., Grabnar M., Stubbs S. L..( 2001;). Comparison of toxinotyping and PCR ribotyping of Clostridium difficile strains and description of novel toxinotypes. Microbiology147:439–447[PubMed]
    [Google Scholar]
  34. Rupnik M., Wilcox M. H., Gerding D. N..( 2009;). Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol7:526–536 [CrossRef][PubMed]
    [Google Scholar]
  35. Schwan C., Stecher B., Tzivelekidis T., van Ham M., Rohde M., Hardt W. D., Wehland J., Aktories K..( 2009;). Clostridium difficile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog5:e1000626 [CrossRef][PubMed]
    [Google Scholar]
  36. Sciara G., Kendrew S. G., Miele A. E., Marsh N. G., Federici L., Malatesta F., Schimperna G., Savino C., Vallone B..( 2003;). The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis. EMBO J22:205–215 [CrossRef][PubMed]
    [Google Scholar]
  37. Sebaihia M., Wren B. W., Mullany P., Fairweather N. F., Minton N., Stabler R., Thomson N. R., Roberts A. P., Cerdeño-Tárraga A. M. et al.& other authors ( 2006;). The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet38:779–786 [CrossRef][PubMed]
    [Google Scholar]
  38. Söding J..( 2005;). Protein homology detection by HMM–HMM comparison. Bioinformatics21:951–960 [CrossRef][PubMed]
    [Google Scholar]
  39. Spigaglia P., Mastrantonio P..( 2002;). Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol40:3470–3475 [CrossRef][PubMed]
    [Google Scholar]
  40. Stabler R. A., Gerding D. N., Songer J. G., Drudy D., Brazier J. S., Trinh H. T., Witney A. A., Hinds J., Wren B. W..( 2006;). Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains. J Bacteriol188:7297–7305 [CrossRef][PubMed]
    [Google Scholar]
  41. Stabler R. A., He M., Dawson L., Martin M., Valiente E., Corton C., Lawley T. D., Sebaihia M., Quail M. A. et al.& other authors ( 2009;). Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol10:R102 [CrossRef][PubMed]
    [Google Scholar]
  42. Voth D. E., Ballard J. D..( 2005;). Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev18:247–263 [CrossRef][PubMed]
    [Google Scholar]
  43. Warny M., Pepin J., Fang A., Killgore G., Thompson A., Brazier J., Frost E., McDonald L. C..( 2005;). Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet366:1079–1084 [CrossRef][PubMed]
    [Google Scholar]
  44. Weese J. S., Wakeford T., Reid-Smith R., Rousseau J., Friendship R..( 2010;). Longitudinal investigation of Clostridium difficile shedding in piglets. Anaerobe16:501–504 [CrossRef][PubMed]
    [Google Scholar]
  45. Zaiss N. H., Rupnik M., Kuijper E. J., Harmanus C., Michielsen D., Janssens K., Nübel U..( 2009;). Typing Clostridium difficile strains based on tandem repeat sequences. BMC Microbiol9:6 [CrossRef][PubMed]
    [Google Scholar]
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