Subtyping of Clostridium difficile PCR ribotype 001 by REP-PCR and PFGE

Abstract

The REP-PCR (repetitive sequence-based PCR using repetitive extragenic palindromic primers) typing method and a modified PFGE method were applied to isolates of Clostridium difficile PCR ribotype 001 with the aim of comparing their performance as methods of subtyping this organism. Of 200 isolates from 60 hospitals tested by REP-PCR, eight subtypes were identified and labelled as REP-PCR subtypes 001-008. The predominant subtype, REP-PCR subtype 003, accounted for 47 % of the total. Fifty-two of the 200 isolates were analysed by a modified PFGE method and seven subtypes were identified, labelled as PF-A-PF-G. There was excellent correlation between REP-PCR subtypes and PFGE subtypes with both methods displaying broadly similar discriminatory powers. However, REP-PCR subtyping proved to be a much easier, cheaper and more rapid method suitable for application for routine subtyping of C. difficile ribotype 001. Application of REP-PCR subtyping to UK isolates of C. difficile PCR ribotype 001 from 60 different centres revealed a wide distribution of REP-PCR subtype 003 throughout England and Wales, with a regional clustering of REP-PCR subtype 001 around Northwest England and North Wales. Analysis of isolates from a single hospital over a 4-year period revealed a change in predominant subtype over time. INTRODUCTIONClostridium difficile is recognized as the major cause of nosocomially acquired antibiotic-associated diarrhoea and pseudomembranous colitis (Bartlett, 1994;George et al., 1978;Tabaqchali & Jumaa, 1995) and is a significant financial burden on modern healthcare resources (Wilcox et al., 1996). Numerous C. difficile reservoirs exist within hospitals, including environmental surfaces, ward and surgical staff (Verity et al., 2001) and colonized new admissions (Clabots et al., 1992). This huge potential for infection of patients from diverse sources necessitates methods for typing C. difficile to understand the epidemiology of outbreaks and isolated cases, to identify any possible incidence of crossinfection and to set up surveillance programmes to monitor virulent strain emergence and hospital reservoirs.Investigations performed at the Anaerobe Reference Laboratory (ARL) in Cardiff revealed that one strain, C. difficile PCR ribotype 001, accounted for 58 % of all hospital isolates tested (Brazier, 2001). This type appears to be endemic throughout most UK hospitals and is associated with both acute and prolonged outbreaks (Brazier & Borriello, 2000), causing serious outbreaks in the UK (Cartmill et al., 1994) and overseas (al-Barrak et al., 1999;Kato et al., 2001; Rafferty et al., 1998).A subtyping study by Fawley et al. (2003) combined RAPD analysis, ribospacer PCR (RS-PCR) and PFGE to identify two subgroups within a small subset of PCR ribotype 001. Another study using pyrolysis mass spectrometry (PMS) identified nine major groups within ribotype 001 (Al-Saif et al., 1998). However, they reported the inability of the PMS method to assign permanent types and the disadvantage of being unable to compare resul...

Molecular Analysis of Clostridium difficile PCR Ribotype 027 Isolates from Eastern and Western Canada

“…REP-PCR has recently been put forth as a useful corollary or subtyping methodology for PCR ribotype 001 strains, offering improved strain type resolution over PCR-ribotyping alone (16,20). While PCR-ribotyping and REP-PCR are both based on the differential amplification of bacterial genomic sequences, their coverage and basis for discrimination are fundamentally different.…”

“…REP-PCR was conducted using the same basic reaction volumes and composition as described above for PCR-ribotyping. Heterogenous primers were generated as described previously by Rahmati et al, but thermocycler conditions were modified from previously published reports (16,20). The modified profile is as follows: an initial denaturation cycle of 2 min at 95°C and 3 s at 94°C, followed by 35 cycles of 30 s at 92°C, 1 min at 40°C, and 1 min at 65°C, with a final extension cycle of 8 min at 65°C.…”

Correlation of Disease Severity with Fecal Toxin Levels in Patients with Clostridium difficile -Associated Diarrhea and Distribution of PCR Ribotypes and Toxin Yields In Vitro of Corresponding Isolates

“…Several typing methods for C. difficile have been developed, and PCR ribotyping, pulsed-field gel electrophoresis, and immunoblot typing offer a high discriminatory power compared to serotyping, but a direct comparison between all methods or systematic combinations of these has to our knowledge not yet been performed (1,4,19,32,33). Further typing of the dominating PCR ribotype in United Kingdom hospitals (UK001, serogroup G) revealed several subtypes (9,10,28). Thus, combinations of typing methods or new methods for typing or subtyping are needed to better define the epidemiology of distinct C. difficile strains and for studies of their clinical virulence.…”

Genetic characterization of clinical isolates of Clostridium difficile using an optimized RAPD protocol and PCR ribotyping reveals strain diversity between two tertiary referral Trusts in the West Midlands, UK

“…Methods such as multi-locus variable number tandem repeat analysis (MLVA) are now becoming increasingly employed to type strains of C. difficile as they have the capacity to identify greater genotypic variability between isolates of C. difficile and identify 'subtypes' within PCR ribotypes (Killgore et al, 2008;Marsh et al, 2006;van den Berg et al, 2007). Subtyping within PCR ribotypes has also previously been demonstrated using other methods such as PFGE, REA and REP-PCR Northey et al, 2005;Killgore et al, 2008;Fawley et al, 2008;Tanner et al, 2010). Despite this, accessibility, in addition to time and cost restraints, means that MLVA is still not widely utilized.…”