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Journal of Clinical Microbiology, July 2000, p. 2791-2792, Vol. 38, No. 7
Pulsed-Field Gel Electrophoresis of Degradation-Sensitive DNAs
from Clostridium difficile PCR Ribotype 1 Strains
Received 3 April 2000/Accepted 3 May 2000
Clostridium difficile is a causative agent of
nosocomially acquired antibiotic-associated diarrhea and
pseudomembranous colitis (3). Typing schemes include
serogrouping, pyrolysis mass spectroscopy, restriction endonuclease
analysis, whole-cell protein profiling, arbitrarily primed PCR,
pulsed-field gel electrophoresis (PFGE), and PCR ribotyping
(2). PFGE is a highly discriminatory method, but a number of
strains of C. difficile are reported to be untypeable due to
degradation of DNA during the procedure (4, 5). Such strains
can be typed by other methods and have been assigned predominantly to
PCR ribotype 1 and serogroup G (4, 5, 7). In the United Kingdom, PCR ribotype 1 accounts for 57% of isolates from hospitalized patients and is endemic in 33 of 58 hospitals surveyed (2). To date all PCR ribotype 1 isolates tested by the Anaerobe Reference Laboratory (Cardiff, United Kingdom) have been untypeable by PFGE; therefore, a method capable of enhanced discrimination of this prevalent ribotype would improve the epidemiology of C. difficile. The aim of this study was to develop a refined PFGE
method capable of analyzing previously untypeable strains.
The reasons for the degradation of DNA in certain strains of C. difficile during PFGE are unclear. Traditionally, proteinase K has
been used to inactivate nucleases liberated after cell lysis, and our
initial, unsuccessful efforts were directed toward protecting DNA
during, and after, cell wall removal. Protocols included increased EDTA
concentration in suspension buffers, heat shock at 70°C, and the
addition of diethylpyrocarbonate to lysozyme-induced spheroplasts prior
to the addition of sodium dodecyl sulfate (1). Recently, Römling and Tümmler (10) documented the
protection of degradation-sensitive DNA in Pseudomonas
aeruginosa during PFGE by the addition of thiourea to Tris buffer.
Thiourea is thought to neutralize a nucleolytic peracid derivative of
Tris that is formed at the anode during electrophoresis (9).
We have evaluated the effect of thiourea in PFGE on a small number of
C. difficile isolates that had been analyzed previously by
PCR ribotyping (8). Five PCR ribotype 1 isolates (one
isolate from the United States and four isolates from disparate
hospitals in the United Kingdom) and single representatives of
ribotypes 2, 10, 15, and 23 were examined. Our standard preparative
PFGE method was used (6), and SmaI fragments were
separated in 1.0% agarose with an initial pulse of 3 s that
increased linearly to 30 s after 20 h. Electrophoresis was
performed with and without thiourea (50 µM) in the running buffer
(Fig. 1).
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FIG. 1.
PFGE patterns obtained with SmaI-digested DNA
from C. difficile isolates of known PCR ribotype.
Electrophoresis was performed in the absence (a) and in the presence
(b) of 50 µM thiourea. Lanes 1 to 5, five degradation-sensitive PCR
ribotype 1 isolates (from the United States and Newcastle, Wrexham,
Tooting, and Truro, United Kingdom, respectively); lane 6, ribotype 2 isolate (Wrexham); lane 7, ribotype 10 isolate (Manchester, United
Kingdom); lane 8, ribotype 15 isolate (Carshalton, United Kingdom);
lane 9, ribotype 23 isolate (Jersey, United Kingdom); lane 10, 
DNA standard (48.5-kbp concatemer).
Genomic DNA was degraded in all PCR ribotype 1 isolates in the absence of thiourea (Fig. 1a), while band patterns were observed when thiourea was present (Fig. 1b). Initial results with the limited sample set seem to suggest that discrimination (subtyping) of isolates belonging to ribotype 1 may be possible since PFGE profiles differ by a significant number of bands (11). The results indicate that addition of thiourea may permit previously untypeable isolates, belonging to the most common PCR ribotype, to be examined by PFGE. Hopefully, this method will facilitate international comparison of typing results and permit genotype subdivision, leading to improved epidemiology.
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FOOTNOTES |
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* Phone: 44 151 706 4410, ext. 4421
Fax: 44 151 706 5849
E-mail: jecmm{at}liverpool.ac.uk.
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John Edward Corkill* | |||||
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Robert Graham Charles Anthony Hart Royal Liverpool University Hospital Department of Medical Microbiology Prescot Street Liverpool L7 8XP Merseyside, United Kingdom | |||||
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Simon Stubbs Anaerobe Reference Unit Public Health Laboratory University Hospital of Wales Heath Park Cardiff CF4 4XW, United Kingdom |
Journal of Clinical Microbiology, July 2000, p. 2791-2792, Vol. 38, No. 7
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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