PCR coupled with mass-spectrometry for detection of Clostridium difficile virulence markers during the emergence of ribotype 027 in Bucharest area

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In recent years Clostridium difficile infection (CDI) has represented a serious public health issue, mainly due to the global spread of the hypervirulent strain NAP1/027/BI. The purpose of the present study was to evaluate the utility of a PCR coupled with electrospray ionization mass spectrometry (ESI-MS) commercial assay for the detection of C. difficile virulence markers. Non-duplicative C. difficile isolates from patients with CDI diagnosed in a tertiary level hospital from Bucharest were tested for toxin A, toxin B, binary toxin genes and deletion in tcdC gene using PCR/capillary gel electrophoresis and PCR/ESI-MS. The study analysed 45 non-duplicative isolates, 33 strains (73.3%) belonging to ribotype 027. The concordance between PCR/capillary gel electrophoresis and PCR/ESI-MS was 100% for toxin A gene, 97.8% for toxin B gene, 91.1% for binary toxin subunit A gene and 95.6% for binary toxin subunit B gene. The general concordance for the complete panel of markers was 88.9% but was 100% for ribotype 027 isolates. PCR/ESI-MS might be a valid method for the detection of C. difficile virulence markers, including binary toxin.

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  • 1. Bauer MP Notermans DW van Benthem BH Brazier JS Wilcox MH Rupnik M et al. Clostridium difficile infection in Europe: a hospital-based survey. Lancet. 2011;377:63-73. DOI: 10.1016/S0140-6736(10)61266-4

  • 2. Vohra P Poxton IR. Comparison of toxin and spore production in clinically relevant strains of Clostridium difficile. Microbiology. 2011;157:1343-53. DOI: 10.1099/mic.0.046243-0

  • 3. Goorhuis A Bakker D Corver J Debast SB Harmanus C Notermans DW et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain polymerase chain reaction ribotype 078. Clin Infect Dis. 2008;47:1162-70. DOI: 10.1086/592257

  • 4. Stabler RA He M Dawson LFT Martin M Valiente E Parkhill J et al Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol. 2009;10:R102. DOI: 10.1186/gb-2009-10-9-r102

  • 5. Lim SK Stuart RL Mackin KE Carter GP Kotsanas D Francis MJ et al. Emergence of a ribotype 244 strain of Clostridium difficile associated with severe disease and related to the epidemic ribotype 027 strain. Clin Infect Dis. 2014;58:1723-30. DOI: 10.1093/cid/ciu203

  • 6. Rafila A Indra A Popescu GA Wewalka G Allerberger F Benea S et al. Occurrence of Clostridium difficile infections due to PCR ribotype 027 in Bucharest Romania. J Infect Dev Ctries. 2014;8:694-8. DOI: 10.3855/jidc.4435

  • 7. Indra A Huhulescu S Schneeweis M Hasenberger P Kernbichler S Fiedler A et al. Characterization of Clostridium difficile isolates using capillary gel electrophoresis –based PCR ribotyping. J Med Microbiol. 2008;57:1377-82. DOI: 10.1099/jmm.0.47714-0

  • 8. van den Berg RJ Claas ECJ Oyib DH Klaassen CHW Dijkshoorn L Brazier JS et al. Characterization of toxin A-negative toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping. J Clin Microbiol. 2004;42:1035–41. DOI: 10.1128/JCM.42.3.1035-1041.2004

  • 9. Kato N Ou CY Kato H Bartley SL Brown VK Dowell VRJ Jr et al. Identification of toxigenic Clostridium difficile by the polymerase chain reaction. J Clin Microbiol. 1991;29:33–7.

  • 10. Kato H Kato N Katow S Maegawa T Nakamura S Lyerly DM. Deletions in the repeating sequences of the toxin A gene of toxin A-negative toxin B-positive Clostridium difficile strains. FEMS Microbiol Lett. 1999;175:197–203. DOI: 10.1111/j.1574-6968.1999. tb13620.x

  • 11. Stubbs SL Brazier JS Talbot PR Duerden BI. PCR-restriction fragment length polymorphism analysis for identification of Bacteroides spp. and characterization of nitroimidazole resistance genes. J Clin Microbiol. 2000;38:3209–13.

  • 12. Spigaglia P Mastrantonio P. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol. 2002;40:3470–5. DOI: 10.1128/JCM.40.9.3470-3475.2002

  • 13. Curry SR Marsh JW Muto CA O’Leary MM Pasculle AW Harrison LH. tcdC Genotypes associated with severe tcdC truncation in an epidemic clone and other strains of Clostridium difficile. J Clin Microbiol. 2007;45:215–21. DOI: 10.1128/JCM.01599-06

  • 14. Wolff D Brüning T Gerritzen A. Rapid detection of the Clostridium difficile ribotype 027 tcdC gene frame shift mutation at position 117 by real-time PCR and melt curve analysis. Eur J Clin Microbiol Infect Dis. 2009;28:959-62. DOI: 10.1007/s10096-009-0731-7

  • 15. Ecker DJ Sampath R Blyn LB Eshoo MW Ivy C Ecker JA et al. Rapid identification and strain-typing of respiratory pathogens for epidemic surveillance. Proc Natl Acad Sci U S A. 2005;102:8012-7. DOI: 10.1073/pnas.0409920102

  • 16. Wolk DM Kaleta EJ Wysocki VH. PCR-electrospray ionization mass spectrometry: the potential to change infectious disease diagnostics in clinical and public health laboratories. J Mol Diagn. 2012;14:295-304. DOI: 10.1016/j.jmoldx.2012.02.005

  • 17. Wilcox MH Shetty N Fawley WN Shemko M Coen P Birtles A et al. Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England. Clin Infect Dis. 2012;55:1056-63. DOI: 10.1093/cid/cis614

  • 18. Popescu G Serban R Pistol A Niculcea A Preda A Lemeni D et al. Clinical and microbiological characterization of Clostridium difficile infection in Romania (2013-2014); a hospital-based study BMC Infect Dis. 2014;14(Suppl 7):O24. DOI: 10.1186/1471-2334-14-S7-O24

  • 19. Drudy D Fanning S Kyne L. Toxin A-negative toxin B-positive Clostridium difficile. Int J Infect Dis. 2007;11:5–10. DOI: 10.1016/j.ijid.2006.04.003

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