Journal of Hospital Infection (2005) 61, 231236

www.elsevierhealth.com/journals/jhin

A modied pulsed-eld gel electrophoresis (PFGE) protocol for subtyping previously non-PFGE typeable isolates of Clostridium difcile polymerase chain reaction ribotype 001
M. Gal, G. Northey, J.S. Brazier*
Anaerobe Reference Laboratory, NPHS Microbiology Cardiff, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK
Received 5 January 2005; accepted 18 January 2005 Available online 5 July 2005

KEYWORDS
Clostridium difcile; Subtyping; PFGE

Summary A modied pulsed-eld gel electrophoresis (PFGE) protocol was developed and applied to 50 isolates of the UK epidemic strain of Clostridium difcile, polymerase chain reaction (PCR) ribotype 001, to develop a PFGE-based subtyping scheme. This protocol overcame the inherent DNA degradation problems associated with typing this strain of C. difcile by this method, and whole genomic digestion with SmaI restriction enzyme yielded seven distinct and reproducible PFGE banding patterns. Modied PFGE is an appropriate method for subtyping C. difcile PCR ribotype 001 that could be used to improve epidemiological investigations. Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. particularly virulent strains. The epidemiology of C. difcile infections in UK hospitals has been monitored at the Anaerobe Reference Laboratory (ARL) at the University Hospital of Wales in Cardiff since 1995 using polymerase chain reaction (PCR) ribotyping.1 This surveillance revealed that C. difcile PCR ribotype 001 was present in over 50% of hospitals in the UK.1 As PCR ribotype 001 is so widespread, attempts to understand the epidemiology of infection at a local level where this type predominates has sometimes been unsuccessful.2 The ability to subtype the UK epidemic strain should lead to a better understanding of local C. difcile epidemiology.

Introduction
Clostridium difcile is a well-recognized nosocomial pathogen responsible for antibiotic-associated diarrhoea and pseudomembraneous colitis. Ward outbreaks involving cross-infection are well documented, and typing investigations are an integral part of epidemiological investigations to determine the source and recognition of

* Corresponding author. Tel.: C44 02920 742378; fax: C44 02920 744123. E-mail address: brazier@cardiff.ac.uk

0195-6701/$ - see front matter Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2005.01.017

232 Genotyping schemes applied to C. difcile have included PCR ribotyping, arbitrarily primed PCR, random-amplied polymorphic DNA PCR (RAPD), amplied fragment length polymorphism, repetitive element sequence-based PCR (rep-PCR), multilocus sequence typing, restriction endonuclease analysis and pulsed-eld gel electrophoresis (PFGE).3,4 PFGE has been used extensively for typing of bacterial species, and is often considered to be the gold standard of genomic typing methods owing to its high discriminatory power and reproducibility. However, problems of non-typeability due to genomic DNA degradation of some C. difcile isolates have been extensively reported,59 most notably with PCR ribotype 001,1012 and this has precluded the widespread use of this method. It has been shown that both rep-PCR and PFGE have greater discriminatory powers than PCR ribotyping as molecular typing methods for C. difcile.9,13 In a further study by Fawley et al.,14 the existence of subtypes within C. difcile PCR ribotype 001 was described. In this small study, 12 local isolates of C. difcile ribotype 001 were examined by PFGE, RAPD and ribospacer PCR (RS-PCR) and this revealed two subtypes. Research performed in our own institution found RAPD to be an unsuitable subtyping method for ribotype 001 due to a lack of reproducibility despite extensive experimental modications (Gemma Northey, ARL, personal communication). The aim of this study was to develop a PFGE protocol that could be used reliably for the subtyping of C. difcile PCR ribotype 001 and to apply PFGE to investigate genomic diversity within this predominant UK strain.

M. Gal et al.
Table I Pulsed-eld gel electrophoresis characterization of 50 Clostridium difcile polymerase chain reaction ribotype 001 strains isolated from patients in UK hospitals Reference number R15202 R7471 R12793 R8366 R14199 R14200 R14201 R14203 R14204 R14205 R14206 R15298 R18908 R19157 R16510 R13050 R7537 R8815 R10095 R10100 R10102 R13929 R12478 R12479 R17309 R10188 R4697 R9758 R9760 R10096 R10490 R10491 R10492 R10498 R10593 R10806 R11162 R11729 R13040 R14202 R16762 R17011 R17100 R18162 R18307 R18769 R19020 R10649 R11581 R12103 Origin Manchester Manchester Bristol Wrexham Chester Chester Chester Chester Chester Chester Chester Bangor Rhyl Preston Nuneaton Leicester Carshalton Carshalton Carshalton Carshalton Carshalton Antrim Bristol Bristol Kent Airedale Carshalton Carshalton Carshalton Carshalton Bridgend Bridgend Bridgend Bridgend Birmingham Dorchester Grantham Nottingham Leicester Chester Birmingham Paisley Cambridge Newport Manchester Lewisham Trafford Manchester Carlisle Ealing Date isolated 2001 1994 1999 1995 2000 2000 2000 2000 2000 2000 2000 2001 2003 2004 2002 1999 1995 1995 1996 1996 1996 2000 1998 1998 2002 1996 1997 1996 1996 1996 1997 1997 1997 1997 1997 1997 1997 1998 1999 2000 2002 2002 2002 2003 2003 2003 2003 1997 1997 1998 PF type A B B C C C C C C C C C C C D D F F F F F F F F F E E E E E E E E E E E E E E E E E E E E E E G G G

Materials and methods

C. difcile isolates
A total of 50 C. difcile PCR ribotype 001 isolates from symptomatic hospital patients from 27 geographically distinct UK hospitals were selected (Table I). The isolates had previously been conrmed as belonging to ribotype 001 by PCR ribotyping performed at the ARL.1 All isolates of C. difcile had been stored frozen at K80 8C on Microbank beads (ProLab Diagnostics, Wirral, UK) and were cultured on Fastidious Anaerobe Agar (FAA; Lab M, Bury, UK) supplemented with 6% horse blood in an anaerobic atmosphere (CO2 10%, H2 10%, N2 80% [v/v]) at 37 8C overnight.

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DNA preparation
Cells were harvested from overnight cultures on FAA plates as opposed to broths, therefore avoiding possible contamination problems. Approximately 10 colonies of C. difcile were harvested from an FAA plate and cells were suspended in 1 mL of PIV buffer [10 mM Tris (pH 8.0), 2 M NaCl2] containing 4% formaldehyde and incubated on ice for 1 h. Cells were harvested by low-speed centrifugation (1000!g, 3 min) and washed three times with 1 mL of cold PIV buffer at 4 8C to remove formaldehyde. The Optical Density (OD) was adjusted to McFarland standard 5.0 and 1 mL of the adjusted cell suspension was used. Cells were collected by low-speed centrifugation and suspended in 100 mL of freshly prepared, lter-sterilized, doublestrength lysis buffer [12 mM Tris-HCl (pH 7.5), 2 M NaCl2, 200 mM EDTA (pH 8.0), 1% Brij-58, 0.4% sodium deoxycholate, 1% sodium lauryl sarcosine, lysozyme 4 mg/mL, RNase 100 mg/mL, mutanolysin 40 U/mL]. Two percent (wt/vol) low-melting-temperature agarose (Bio-Rad Laboratories, Hertfordshire, UK) previously heated to 50 8C was added immediately. The cell suspension was mixed gently and pipetted into plug moulds (Bio-Rad) that were left to set at 4 8C for 10 min. The agarose plugs were expelled into 2 mL of 1!lysis buffer and incubated overnight at 37 8C with gentle agitation. The lysis buffer was removed and replaced with 1 mL of freshly prepared, lter-sterilized ESP buffer [0.5 M EDTA (pH 8.0), 10% sodium lauryl sarcosine, 200 mg/mL proteinaseK] that had been prewarmed to 50 8C for 1 h. Incubation proceeded at 50 8C for 6 h without agitation. The ESP buffer was removed and replaced with 1 mL of fresh ESP buffer and the incubation continued overnight. Next day, the ESP buffer was replaced once more and the incubation was continued for a further 6 h. The incubation temperature was maintained at 50 8C using prewarmed buffer throughout. Plugs were washed twice in 3 mL of T10E1 buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)]. The buffer was replaced by 1 mL of T10E1 buffer containing 1 mM phenylmethylsulphonyl uoride (stock 100 mM in isopropanol) and the plugs were incubated at 40 8C for 1 h to inactivate the proteinaseK. The buffer was removed and the plugs were washed three more times with 3 mL T10E1 buffer for 30 min. Plugs were stored in T10E1 at 4 8C for up to seven days. Prior to enzyme digestion, the EDTA concentration of the TE buffer was lowered [10 mM Tris-HCl 0.1 mM EDTA (pH 8.0)] and the plugs were washed three times for 60 min in this buffer.

Restriction digestion of DNA

Plugs were cut into 5-mm squares using a sterile cover slip and equilibrated at 4 8C for 1 h in 200 mL of 1!SmaI restriction buffer. The restriction buffer was replaced with fresh 1!restriction buffer and 40 U of SmaI enzyme per plug (New England Biolabs, Herts, UK) in a total volume of 100 mL and incubated at 25 8C for 2 h. To ensure complete restriction digestion, a further 10 U of SmaI was added to each plug and incubation was continued for a further 2 h. Following digestion, plugs were loaded into a 1% PFGE-grade agarose gel (Bio-Rad). Tris-borateEDTA 0.5![45 mM Tris-HCl, 46 mM boric acid, 1 mM EDTA (pH 8.0)] buffer and 200 mM thiourea was used for gel preparation and for the running buffer.

PFGE
The type strain of C. difcile PCR ribotype 001 (R8366) was used as the reference control strain and molecular marker in each case. Gels were run for 24 h at 14 8C using a CHEF DR-III system (Bio-Rad Laboratories) at 5.5 V/cm with initial and nal pulse times of 5 s and 60 s, respectively. Following electrophoresis, the gel was stained for 1 h in distilled water containing 0.5 mg/mL ethidium bromide. Gels were destained in distilled water for 1 h. The DNA bands were visualized under ultraviolet illumination and the gel image was captured and digitized as tif les using the Gel-Doc 2000 system (Bio-Rad).

Fingerprint analysis
The macrorestriction patterns were analysed using GelComparII (Applied Maths, Kortrijk, Belgium). Gels were normalized and aligned by associating the R8366 strain bands as a reference pattern on each gel. The similarity coefcient between the PFGE restriction patterns was determined and clustered by the unweighted pair group method with arithmetic averages. Amplication products were scored as present or absent without regard to the mode of inheritance. New types were assigned based on single band differences.

Results and discussion

In our hands, none of the previously published PFGE methods for Clostridium spp.12,13,1517 were found to achieve consistently satisfactory results with C. difcile ribotype 001. Problems were encountered

234 with reproducibility, incomplete cell lysis, DNA degradation and poor fragment denition. Through experimentation, a number of crucial steps were identied during the optimization of the PFGE protocol. Lysis of bacterial cells and subsequent DNA yields improved with the following modications: increasing lysozyme concentrations and lysis incubation times; using mutanolysion in addition to lysozyme; and suspending the cells in lysis buffer rather than wash buffer prior to addition of agarose. Increasing the duration and concentration of proteinaseK treatment was found to be necessary to reduce smearing and DNA degradation. Formaldehyde xation of cells on ice produced sharper bands and facilitated easier band interpretation. The addition of thiourea to the Tris running buffer was found to be essential. Overnight incubation of the DNA with SmaI was not necessary as a 4 h period was sufcient to achieve complete restriction digestion. Occasionally, restriction digestion of DNA was incomplete, resulting in the presence of additional higher molecular weight bands, thus making computer-assisted analysis problematic. On addition of a further 10 U of SmaI restriction enzyme for the last 2 h of incubation, digestion was carried out to completion and the additional bands subsequently disappeared. These modications gave reproducible and discriminatory results, with all isolates of C. difcile PCR ribotype 001 being typeable.

M. Gal et al. Electrophoresis of SmaI-digested DNA yielded well-resolved patterns of between nine and eleven analysable fragments that ranged in size between approximately 2.2 mb and 285 kb (Figure 1). Analysis of band patterns produced by PFGE is relatively simple and can be done using a number of commercially available packages including GelComparII (Applied Maths). Using this software package, a library of restriction types was created and used as a reference to which any new strain was compared. Seven different PFGE types (PF) were observed with all isolates sharing ve common bands. Twenty-two of the 50 isolates with identical patterns were designated PF-E and had been isolated from hospital patients around the UK over a nine-year period. The second and third largest PF groups (PF-C and PF-F) were composed of eleven and nine isolates respectively, with PF-C isolates being clustered in the North of England and PF-F being found to be dispersed throughout the UK. The three smaller groups (PF-B, PF-D and PF-G) contained two, two and three isolates, respectively. One isolate (PF-A) yielded a unique prole. Although the numbers used in this study are too small for a comprehensive UK epidemiological investigation, the initial results indicate that extending this work might prove valuable. The dendrogram obtained from numerical analysis of the SmaI restriction PFGE proles is shown in Figure 2. The analysis revealed genomic diversity

Figure 1 Pulsed-eld gel electrophoresis of Clostridium difcile ribotype 001 strains showing the seven different PF types. Lanes 1, 8 and 12ZPF-C; lane 2ZPF-A; lane 3ZPF-B; lane 4ZPF-D; lanes 5, 10 and 11ZPF-E; lanes 6 and 9ZPFF; lane 7ZPF-G; lane 9ZPF-F. PF-C was used as the control and ladder for normalization in each gel.

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Figure 2 Dendrogram for isolates digested with SmaI after cluster analysis. Strain reference numbers and the sources are shown in the vertical axis. The numbers on the horizontal axis indicate the percent similarities as determined with the unweighted pair group method.

with similarity coefcients ranging from 70% to 93%. According to the principles of Tenover et al.,18 strain relatedness can be determined by comparing the number and position of bands. It was proposed that isolates with the same pattern are considered to be the same strain. One to three band differences would be attributable to a single genetic event and these strains could be considered to be closely related. Differences of between two and four bands could be due to two separate genetic events and these strains may possibly be related. However, it has been demonstrated that a single genetic event such as the insertion of an insertion sequence (IS) sequence can produce up to seven band differences.19 Therefore, these criteria of relatedness mainly apply to localized outbreaks, and application of these principles to temporally unrelated strains is probably limited. On initial investigation, we found that the PFGE banding patterns produced by isolates of C. difcile ribotype 001 were more similar to one another than those produced by other C. difcile ribotypes, indicating a higher degree of relatedness (data not shown). A disadvantage of the PFGE method as a routine typing method is low throughput due to the length of the procedure in comparison with PCR-based genotyping methods, taking on average four to eight days to complete. Various shorter protocols have been developed for PFGE of Clostridium spp.;5,17,20 however, our experience was that these shortened protocols were unsuccessful for C. difcile PCR ribotype 001. Other methods of subtyping C. difcile PCR ribotype 001 have been described including RS-PCR and RAPD. However, despite extensive attempts to optimize RAPD in our laboratory, this method was found to be unsuitable owing to non-reproducibility both within and between batches (Gemma Northey, personal communication). We also found that RS-PCR could not discriminate between the seven PFGE types identied from PCR ribotype 001 (data not shown). This

is not surprising as both RS-PCR and PCR ribotyping primers target the same conserved regions within the 16S and 23S rDNA anking the intergenic spacer region. In conclusion, our modied PFGE protocol was found to be highly reproducible and could distinguish seven distinct PF subtypes in a subset of 50 geographically diverse isolates of C. difcile PCR ribotype 001. Further application of this method to geographically distinct isolates may promote our understanding of the epidemiology of UK C. difcile infections. However, PFGE still has disadvantages in terms of its complexity, low throughput and high consumable costs, and other methods of subtyping C. difcile PCR ribotype 001 should be investigated.

Acknowledgements
We acknowledge the support given to this work by the late Professor Roger Freeman. We thank Dr. Val Hall, Trefor Morris and Carol Davis for their technical assistance.

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