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Journal of Medical Microbiology (2005), 54, 155–157
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Communication
DOI 10.1099/jmm.0.45808-0
An improved protocol for pulsed-field gel
electrophoresis typing of Clostridium difficile
R. Alonso, A. Martı́n, T. Peláez, M. Marı́n, M. Rodrı́guez-Creixéms and E. Bouza
Correspondence
R. Alonso
Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario ‘‘Gregorio
Marañón’’, C/Doctor Esquerdo, 46, 28007 Madrid, Spain
ralonso.hgugm@salud.
madrid.org
Received 2 July 2004
Accepted 24 October 2004
Pulsed-field gel electrophoresis (PFGE) is the ‘gold standard’ technique for bacterial typing and has
proved to be discriminatory and reproducible for typing Clostridium difficile. Nevertheless, a high
proportion of strains are non-typable by this technique due to the degradation of the DNA during the
process. The introduction of several modifications in the PFGE standard procedure increased
typability from 40 % (90 isolates) to 100 % (220 isolates) while maintaining the high degree of
discrimination and reproducibility of the technique.
Introduction
Standard protocol. Briefly, the recommended standard protocol was
Clostridium difficile is the main aetiologic agent of nosocomial diarrhoea (Barbut et al., 1996) and is responsible for an
important increase in hospital stays, with high healthcare and
economic repercussions (Wilcox et al., 1996).
as follows. A bacterial culture was inoculated into LB broth with
agitation and grown to an OD600 of 0.8–1.0. Then 5 3 108 bacteria
were removed for each millilitre of agarose plug to be made and
centrifuged to obtain the bacterial pellet. The supernatant was discarded
and the pellet resuspended in 500 ìl of cell suspension buffer. The
bacterial suspension was warmed to 50 8C.
Typing of the micro-organism can be useful in the detection
and control of epidemic outbreaks and endemic situations,
and in the characterization of recurrences (Alonso et al.,
2001; Kato et al., 1996). Different approaches have been used
for typing bacteria, although PFGE has traditionally been the
gold standard (Finney, 1993). PFGE has proved to be
discriminatory and reproducible for typing C. difficile; however, a considerable proportion of strains are non-typable by
this technique due to degradation of the DNA during the
procedure, making uninterpretable gel smears (Kato et al.,
1994; Bidet et al., 2000). Different modifications of the typing
procedure have been proposed (Corkill et al., 2000), but they
have led to variable results with only partial improvement
(Klaassen et al., 2002; Fawley & Wilcox, 2002).
The aim of this study was to develop a new PFGE protocol
which can offer universal typability for all our C. difficile
strains.
Methods
Samples and tests. Two hundred and twenty toxigenic C. difficile
isolates were included in this study. We obtained isolates from the
diarrhoeic stool samples of patients admitted to our hospital over a
period of 6 months. Cultures, identification and toxin detection were
carried out using standard procedures. The CHEF bacterial DNA plug
kit (BIO-RAD) was used for PFGE typing the isolates. The standard
manufacturer’s recommended technique and our modified protocol
were performed and compared.
The 2 % CleanCut agarose was melted and equilibrated to 50 8C. Five
hundred millilitres of agarose was added to the bacterial suspension and
mixed thoroughly. The mixture was transferred to plug moulds and the
agarose was allowed to solidify at 4 8C for 10–15 min.
The solidified plugs were incubated in 1 mg ml 1 lysozyme solution
(100 ìl 25 mg ml 1 lysozyme stock plus 2.5 ml lysozyme buffer) for 2 h
at 37 8C. The lysozyme was removed and the plugs were rinsed with
sterile water. The plugs were incubated with .20 U ml 1 proteinase K
solution (100 ìl of .600 U ml 1 proteinase K stock in 2.5 ml of
proteinase K reaction buffer) at 50 8C for 24–96 h. The plugs were
washed four times in 13 wash buffer for 1 h each at room temperature
with gentle agitation. The second or third wash contained 1 mM PMSF
to inactivate residual proteinase K.
Prior to endonuclease restriction, each plug was washed for 1 h in 1 ml
0.13 wash buffer and then rinsed with fresh 0.13 wash buffer. The plug
was incubated for 1 h with 1 ml of 13 restriction enzyme buffer at room
temperature and then overnight in 300 ìl of 13 restriction enzyme
buffer containing 30–50 U of the restriction enzyme at the appropriate
temperature. After overnight digestion, the buffer was removed and the
plug was incubated for 30 min in 13 wash buffer and then equilibrated
in the appropriate concentration of gel-running buffer (e.g. 0.53 TBE).
The manufacturer recommended loading one-quarter to one-third of a
plug per well for electrophoresis. Although the manufacturer does not
give any recommendation about the electrophoresis itself, most of the
standard published series use 1 % agarose gels in 0.53 TBE buffer and
the electrophoresis parameters vary widely depending on the restriction
endonuclease used.
Modified protocol. Our protocol was based upon the standard
commercial procedure as described, with the following modifications.
This paper was presented at the First International Clostridium difficile
Symposium, Kranjska Gora, Slovenia, 5–7 May 2004.
Fresh 24 h plate cultures were always used. When unfreezing strains,
they were grown three times in CCFA or Brucella agar before starting.
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R. Alonso and others
BHI tubes were inoculated and incubated for 24 h to obtain a highdensity inoculum (OD600 .1.2). Incubations of more than 24 h were not
performed. We removed 109 bacteria for each millilitre of agarose plug
to be made. A fresh preparation of the BHI culture was observed by
microscopy at 31000 magnification to check for the presence of spores.
Cultures with a high proportion of spores (over 40 %) were ruled out.
Lysozyme and proteinase K (Sigma) were prepared ‘in-house’ and used
at high concentrations (2 mg ml 1 and 75 U ml 1 , respectively). Proteinase K incubation was no longer than 18 h. Washing steps were
reduced to 30 min. Endonuclease digestions were performed with high
concentrations of enzyme (SmaI, 60 U in 300 ìl volumes). Thiourea
(Sigma), always freshly prepared and kept in darkness until use, was
included in the gel and running buffer in high concentrations (200 ìM).
Electrophoresis. Electrophoresis was run at 195 V for 20 h with pulse
times starting at 4 s and ending at 30 s. Gels were stained with ethidium
bromide, visualized under UV transillumination and recorded.
Results and Discussion
Using the standard procedure, almost 60 % of the assayed
isolates (130) remained untyped by PFGE (Fig. 1). The
remaining strains offered good PFGE patterns that were
reproduced on every occasion (e.g. the reference strain, C.
difficile ATCC 9689, was included in every run and was
always correctly typed).
Although most of our non-typable isolates (98, 75 %)
belonged to our most prevalent ribotype (R1), a considerable
proportion (32, 25 %) were from several others (data not
shown). Some other authors have reported the same phenomenon with PFGE and C. difficile at different frequencies.
In most cases, non-typable strains belonged to serogroup G
and ribotype 1 (O’Neill et al., 1993; Kato et al., 1996) (some
strains from this serogroup are typable; Bidet et al., 2000),
although others have also reported the problem in different
strains (Kato et al., 2001; Spigaglia et al., 2001). Unfortunately, we did not serotype our strains and we used a different
ribotyping scheme (Bidet et al., 2000), as a result of which we
do not know whether our non-typable strains belong to those
reported major groups.
With the described modifications to the protocol we increased the typability of the technique, enabling all our
C. difficile isolates to be characterized by PFGE (Fig. 1b).
Several factors have been proposed to explain the lack of
typability in C. difficile by PFGE. Firstly, the micro-organism
has very potent endonucleases that may degrade the extracted DNA inside the agarose plug during the extended
process (Spigaglia et al., 2001). Some strains may produce
fewer nucleases, which could be removed or degraded during
nucleic acid extraction. In our proposed protocol, we
shortened incubation and washing times in order to avoid
or reduce such degradation. The high concentrations of
proteinase K used could also help to digest nucleases more
efficiently.
Secondly, it has been reported that a nucleolytic peracid
derivative of Tris may form at the anode during electrophoresis and this chemical nucleolysis could be inhibited by the
addition of thiourea to the running buffer (Ray et al., 1995).
This effect has also been described for other micro-organisms
(e.g. Pseudomonas aeruginosa; Römling & Tümmler, 2000).
The concentration of thiourea is controversial, and while
some authors defend that 50 ìM is enough (Corkill et al.,
2000), others report concentrations of up to 200 ìM in both
the gel and the buffer (Fawley & Wilcox, 2002). We found
that 50 ìM in the buffer had a reduced effect whereas the best
results were achieved with 200 ìM in gel and buffer.
Thirdly, we think it is extremely important to avoid spore
formation. Cultures with a high rate of sporulated forms
probably exhibit poorer lysis and produce less DNA. For this
reason we recommend working with fresh cultures and
screening for the presence of spores by microscopy before
starting the DNA-extraction process. We also recommend
using high lysozyme and proteinase K concentrations to
facilitate spore lysis. Hypothetically, the strains belonging to
our major ribotype could be more likely to produce spores,
which could explain both the difficulty in PFGE typing and a
favourable spread in the hospital. According to this, many
outbreaks have been reported to be caused by PFGE nontypable strains (Kato et al., 2001; Samore et al., 1997).
In conclusion, the above-mentioned modifications enabled
us to achieve complete typability (increasing from 40 % to
100 %) within the assayed isolates while maintaining both a
high degree of discrimination and the reproducibility of the
technique.
Acknowledgements
Fig. 1. Example of PFGE patterns belonging to five representative
clinical isolates of C. difficile using (a) standard procedures and (b) the
modified protocol.
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This paper has been supported in part by ‘Red Española de Investigación
en Patologı́a Infecciosa’ (REIPI – CO3/14) and by ‘Fondo de Investigaciones Sanitarias’ (FIS 02/1049). We thank Thomas O’Boyle for his
help with the correction of the English version of this manuscript.
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Journal of Medical Microbiology 54
Improved protocol for PFGE typing of C. difficile
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