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

Open access


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.

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

Revista Romana de Medicina de Laborator

Romanian Journal of Laboratory Medicine

Journal Information

IMPACT FACTOR 2017: 0.400
5-year IMPACT FACTOR: 0.320

CiteScore 2017: 0.31

SCImago Journal Rank (SJR) 2017: 0.144
Source Normalized Impact per Paper (SNIP) 2017: 0.195

Cited By


All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 113 113 9
PDF Downloads 39 39 6