Abstract
Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson’s index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa (http://www.dlst.org), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.
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References
Freeman J, Bauer MP, Baines SD, Corver J, Fawley WN, Goorhuis B, Kuijper EJ, Wilcox MH (2010) The changing epidemiology of Clostridium difficile infections. Clin Microbiol Rev 23(3):529–549
Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, Dunn JR, Farley MM, Holzbauer SM, Meek JI, Phipps EC, Wilson LE, Winston LG, Cohen JA, Limbago BM, Fridkin SK, Gerding DN, McDonald LC (2015) Burden of Clostridium difficile infection in the United States. N Engl J Med 372(9):825–834
Rupnik M, Wilcox MH, Gerding DN (2009) Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 7(7):526–536
Peniche AG, Savidge TC, Dann SM (2013) Recent insights into Clostridium difficile pathogenesis. Curr Opin Infect Dis 26(5):447–453
Carroll KC, Bartlett JG (2011) Biology of Clostridium difficile: implications for epidemiology and diagnosis. Annu Rev Microbiol 65:501–521
Honda H, Dubberke ER (2014) The changing epidemiology of Clostridium difficile infection. Curr Opin Gastroenterol 30(1):54–62
Eyre DW, Cule ML, Wilson DJ, Griffiths D, Vaughan A, O’Connor L, Ip CL, Golubchik T, Batty EM, Finney JM, Wyllie DH, Didelot X, Piazza P, Bowden R, Dingle KE, Harding RM, Crook DW, Wilcox MH, Peto TE, Walker AS (2013) Diverse sources of C. difficile infection identified on whole-genome sequencing. N Engl J Med 369(13):1195–1205
Mac Aogáin M, Moloney G, Kilkenny S, Kelleher M, Kelleghan M, Boyle B, Rogers TR (2015) Whole-genome sequencing improves discrimination of relapse from reinfection and identifies transmission events among patients with recurrent Clostridium difficile infections. J Hosp Infect 90(2):108–116
Bidet P, Barbut F, Lalande V, Burghoffer B, Petit JC (1999) Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett 175(2):261–266
Aires-de-Sousa M, Boye K, de Lencastre H, Deplano A, Enright MC, Etienne J, Friedrich A, Harmsen D, Holmes A, Huijsdens XW, Kearns AM, Mellmann A, Meugnier H, Rasheed JK, Spalburg E, Strommenger B, Struelens MJ, Tenover FC, Thomas J, Vogel U, Westh H, Xu J, Witte W (2006) High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. J Clin Microbiol 44(2):619–621
Enright MC, Spratt BG (1999) Multilocus sequence typing. Trends Microbiol 7(12):482–487
Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin R, Zhang Q, Zhou J, Zurth K, Caugant DA, Feavers IM, Achtman M, Spratt BG (1998) Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95(6):3140–3145
Kuhn G, Francioli P, Blanc DS (2007) Double-locus sequence typing using clfB and spa, a fast and simple method for epidemiological typing of methicillin-resistant Staphylococcus aureus. J Clin Microbiol 45(1):54–62
Basset P, Blanc DS (2014) Fast and simple epidemiological typing of Pseudomonas aeruginosa using the double-locus sequence typing (DLST) method. Eur J Clin Microbiol Infect Dis 33(6):927–932
Persson S, Torpdahl M, Olsen KE (2008) New multiplex PCR method for the detection of Clostridium difficile toxin A (tcdA) and toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes applied to a Danish strain collection. Clin Microbiol Infect 14(11):1057–1064
Zaiss NH, Rupnik M, Kuijper EJ, Harmanus C, Michielsen D, Janssens K, Nübel U (2009) Typing Clostridium difficile strains based on tandem repeat sequences. BMC Microbiol 9:6
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG (2012) Primer3—new capabilities and interfaces. Nucleic Acids Res 40(15), e115
Griffiths D, Fawley W, Kachrimanidou M, Bowden R, Crook DW, Fung R, Golubchik T, Harding RM, Jeffery KJ, Jolley KA, Kirton R, Peto TE, Rees G, Stoesser N, Vaughan A, Walker AS, Young BC, Wilcox M, Dingle KE (2010) Multilocus sequence typing of Clostridium difficile. J Clin Microbiol 48(3):770–778
Hunter PR (1990) Reproducibility and indices of discriminatory power of microbial typing methods. J Clin Microbiol 28(9):1903–1905
Severiano A, Pinto FR, Ramirez M, Carriço JA (2011) Adjusted Wallace coefficient as a measure of congruence between typing methods. J Clin Microbiol 49(11):3997–4000
Marsh JW, O’Leary MM, Shutt KA, Pasculle AW, Johnson S, Gerding DN, Muto CA, Harrison LH (2006) Multilocus variable-number tandem-repeat analysis for investigation of Clostridium difficile transmission in Hospitals. J Clin Microbiol 44(7):2558–2566
Walker AS, Eyre DW, Wyllie DH, Dingle KE, Harding RM, O’Connor L, Griffiths D, Vaughan A, Finney J, Wilcox MH, Crook DW, Peto TE (2012) Characterisation of Clostridium difficile hospital ward-based transmission using extensive epidemiological data and molecular typing. PLoS Med 9(2), e1001172
Curry SR, Muto CA, Schlackman JL, Pasculle AW, Shutt KA, Marsh JW, Harrison LH (2013) Use of multilocus variable number of tandem repeats analysis genotyping to determine the role of asymptomatic carriers in Clostridium difficile transmission. Clin Infect Dis 57(8):1094–1102
Bidet P, Lalande V, Salauze B, Burghoffer B, Avesani V, Delmée M, Rossier A, Barbut F, Petit JC (2000) Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile. J Clin Microbiol 38(7):2484–2487
Killgore G, Thompson A, Johnson S, Brazier J, Kuijper E, Pepin J, Frost EH, Savelkoul P, Nicholson B, van den Berg RJ, Kato H, Sambol SP, Zukowski W, Woods C, Limbago B, Gerding DN, McDonald LC (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 Microbiol 46(2):431–437
Acknowledgements
We thank F. Barbut (Hôpital Saint-Antoine, 184 rue du Faubourg Saint-Antoine, 75012 Paris, France) for providing us with C. difficile strains with known ribotypes (Supplementary file 1) and C. Choulat for the technical assistance.
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Stojanov, M., Magalhaes, B., Terletsky, V. et al. Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile . Eur J Clin Microbiol Infect Dis 35, 175–181 (2016). https://doi.org/10.1007/s10096-015-2525-4
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DOI: https://doi.org/10.1007/s10096-015-2525-4