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Journal of Clinical Microbiology, July 2002, p. 2452-2458, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2452-2458.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Clostridium difficile Genotyping Based on slpA Variable Region in S-Layer Gene Sequence: an Alternative to Serotyping

Tuomo Karjalainen,1 Nicolas Saumier,1 Marie-Claude Barc,1 Michel Delmée,2 and Anne Collignon1*

Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 92296 ChÂtenay-Malabry Cedex, France,1 Université Catholique de Louvain, Faculté de Médecine, Unité de Microbiologie, Brussels 1200, Belgium2

Received 10 January 2002/ Returned for modification 16 February 2002/ Accepted 13 April 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent investigations of Clostridium difficile cell wall components have revealed the presence of an S-layer encoded by the slpA gene. The aim of this study was to determine whether slpA genotyping can be used as an alternative to serotyping. The variable regions of slpA were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly. Amplified products were analyzed after restriction enzyme digestion and DNA sequencing. The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups. These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the slpA variable region, could constitute an alternative typing method for determining C. difficile serotypes.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Clostridium difficile is the etiological agent of antibiotic-associated pseudomembranous colitis and is considered the major cause of nosocomial diarrhea. Its pathogenicity is mediated by two toxins, A and B, both of which damage the human colonic mucosa and are potent tissue-damaging enzymes (6). Confirmed and putative accessory virulence factors that could play a role in intestinal colonization have been identified, including a capsule (11), proteolytic enzymes (22, 24), and adhesins involved in mucus and cell association (15-17, 26-29, 33).

Many bacteria express a surface-exposed proteinaceous layer, termed the S-layer, that forms a regular two-dimensional array visible by electron microscopy. Previous studies have shown that a high degree of variability exists in the molecular masses of the two proteins composing the C. difficile S-layer (8, 10, 19). Each strain carries a higher-molecular-mass protein of 48 to 56 kDa, encoded by the C-terminal, conserved part of the slpA gene, and a lower-molecular-mass protein of 36 to 45 kDa, coded for by the variable N-terminal part of the gene (10, 17, 19). The lower-molecular-mass S-layer protein, referred to as the P36 protein, appears to be located on the exterior surface of the bacteria and has adhesive properties (10, 17). Interestingly, the gene encoding the S-layer precursor is present in a genetic cluster locus carrying 17 open reading frames (ORFs), 11 of which carry a similar two-domain architecture, likely to encode surface-anchored proteins (17).

Most epidemiological studies of C. difficile have been performed by using several typing systems. Serogrouping by slide agglutination or enzyme-linked immunosorbent assay with rabbit antisera enables the differentiation of 10 major serogroups, which are represented by capital letters (A, B, C, D, F, G, H, I, K, and X) (14, 31). In serogroup A, another 20 subgroups (subgroups A1 to A20) can be distinguished by polyacrylamide gel electrophoresis (14); these subgroups possess serogroup-specific somatic antigens but have a flagellar antigen in common that is responsible for cross-agglutination on slides (12). Recently, new molecular techniques have been developed for C. difficile typing (7, 18).

The aim of this work was to study the genotypic variability of the slpA gene encoding the outwardly exposed domain of the major C. difficile surface protein. Amplicons obtained by PCR from serogroup reference strains and various clinical isolates were analyzed by restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains, media, and growth conditions. Thirty-two C. difficile isolates belonging to 10 different serogroups (A, B, C, D, F, G, H, I, K, and X) were examined. The strains included the 10 serogroup reference strains and clinical isolates (Table 1). Serogroups had been previously determined by slide agglutination with rabbit antisera (13) or enzyme-linked immunosorbent assay and confirmed by typing by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (14). Toxin A production was determined by the C. difficile toxin A test (Oxoid). In vitro cytotoxin (toxin B) determination was performed by using tissue culture cells as previously described (28).


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  		TABLE 1. C. difficile isolates studied and their slpA RFLP patternsa

 
Bacteria were grown under anaerobic conditions in Tryptone-glucose-yeast infusion broth (Difco Laboratories, Detroit, Mich.) for 48 h.

Genomic DNA isolation. DNA was extracted from 10 ml of an overnight anaerobic culture of C. difficile in accordance with the protocol provided in the Puregene DNA gram-positive bacterium and yeast DNA extraction kit (Gentra Systems, Minneapolis, Minn.; www.gentra.com).

PCR amplification of slpA. For amplification of the variable domain of the slpA gene (Fig. 1) from various C. difficile isolates, the primers used were slpAV3 (5'-ATGAATAAGAAAAAYWTAGCAATRGC-3') and slpAV5 (5'-TCTATTCTATCTT CTCCWGCTAC-3'), where Y = CT, W = AT, and R = AG. For amplification of the entire gene, primers slpAV3 and slpAC5 (5'-AGCKATACCTTTACCWACTTG-3'), where K = TG, were used. DNA amplification by PCR was performed in a reaction volume of 50 µl consisting of 1 µl of purified genomic DNA (1 µg/µl), 1 µl each of the 5' and 3' primers at 20 pmol/µl, 25 µl of water, and 25 µl of Ready-Mix Taq PCR Reaction Mix with MgCl2 (Sigma). Initial denaturation was carried out at 95°C for 5 min. Thirty-five cycles of amplification were performed in a Perkin-Elmer GeneAmp PCR system 2400 thermocycler. Each cycle consisted of three steps: denaturation at 95°C (30 s), annealing at 45°C (1 min), and extension at 72°C (1 min to amplify the conserved or variable domain and 2 min to amplify the entire gene). An additional step of extension for 10 min at 72°C was performed at the end of the amplification to complete the extension of the primers. Samples (3 µl) of amplified products were analyzed by electrophoresis in a 1.0% (wt/vol) agarose gel with a 100-bp ladder (Amersham-Pharmacia Biotech) as the molecular size marker.



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  		FIG. 1. Structural organization of the slpA gene of C. difficile. The conserved (C) domain is in gray, and the variable (V) domain is represented by the hatched bar. Positions of the primers used for amplification of the domains are indicated below the gene structures. Gene length is indicated in kilobases above the gene structures.

 
RFLP analysis. Five microliters of PCR-amplified products, purified with the Wizard PCR Preps DNA purification system (Promega), were digested with the restriction enzymes DraI, HinfI, PvuII, and RsaI in accordance with the vendor's (Life Technologies) recommendations. The digested amplified products were analyzed by electrophoresis in a 1.0% (wt/vol) agarose gel.

Sequence analysis. PCR products were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany). DNA sequencing was carried out with the BigDye terminator DNA sequencing kit (PE Applied Biosystems, Warrington, England). The samples were analyzed with an automated DNA sequencer, the ABI PRISM 310 genetic analyzer (Perkin-Elmer). Initial sequencing was carried out with the same primers as used for PCR. More sequence was acquired by using primers the sequences of which were derived from the internal sequence data obtained for the variable region.

Computer analyses. Nucleotide and protein sequence alignments were performed with the DNA CLUSTAL W program (30).

Nucleotide sequence accession numbers. The GenBank nucleotide sequence accession numbers of the slpA variable region from the C. difficile isolates studied are given in Table 1.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PCR amplification and sequence analysis of the slpA variable region from serogroup reference strains. It has been previously demonstrated that the C. difficile slpA gene carries a conserved 3' half and a variable 5' half in the strains studied (Fig. 1) (17). The gene encodes a larger precursor protein that is probably cleaved into distinct S-layer proteins. The protein corresponding to the N-terminal half (referred to as P36) is exposed to the external milieu and thus could play a role in determining serogroup specificity. To investigate this possibility, the variable region of the slpA gene from 10 serogroup reference strains was amplified by PCR with the primers indicated in Fig. 1 (Table 1). A PCR product of approximately 1 kb was obtained from all of the strains (Table 1).

The PCR product was digested with the restriction enzymes DraI, HinfI, PvuII, and RsaI. Each serogroup revealed a different RFLP profile after electrophoresis in an agarose gel of the restriction products (not shown), suggesting that the slpA variable-region sequence is different in each serogroup. For this reason, the slpA gene variable region from the reference strains was sequenced. The length of the variable region varied from 1,017 to 1,185 nucleotides (339 to 395 amino acids) (Table 1). As shown in Fig. 2, alignment of the amino acid sequences of the variable region revealed that each sequence was unique, with interserogroup sequence homology ranging from 11% (between serogroups A and I) to 64% (between serogroups C and F) (Table 2).



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  		FIG.2. Multiple alignments of the predicted amino acid sequences encoded by variants of the variable region of the slpA gene from Delmée's serogroup reference strains A1, B, C, D, F, G, H, I, K, and X. Furthermore, three additional SlpA sequences from serogroup A (Kohn, 9354, and TO005) are included in the alignment. Additional sequence data were obtained for at least two isolates in each serogroup (Table 1). Analysis of the data showed that the slpA gene sequence from strains EX560 and CO109 was 100% identical to that of the serogroup B reference strain; that of strains 630 and C253 was 100% identical to that of the serogroup C reference strain; that of strains 90-111 and 93-136 was 100% identical to that of the serogroup D reference strain; that of strains R7404, GAI95600, and GAI95601 was 100% identical to that of the serogroup F reference strain; that of strains R8366 and 93-392 was 100% identical to that of the serogroup G reference strain; that of strains 89-638 and 90-204 was 100% identical to that of the serogroup H reference strain; that of strains 56026 and 54823 was 100% identical to that of the serogroup I reference strain; that of strains 94-416 and 48-515 was 100% identical to that of the serogroup K reference strain; and that of strains 12-934 and 36-678 was 100% identical to that of the serogroup X reference strain. Identical residues are indicated by asterisks below the alignment; functionally identical residues are indicated by colons or periods.

 

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  		TABLE 2. Pairwise comparisons of amino acid sequence homology of the variable region of the SlpA protein between C. difficile serogroups

 
The presence of nucleotide sequence differences in the slpA genes of C. difficile reference strains allowed the establishment of a precise computer-generated RFLP profile for each serogroup. As shown in Fig. 3 and Table 1, with the exception of serogroup A, unique restriction patterns were produced for each serogroup by digestion with the restriction enzymes HinfI and RsaI. PvuII digestion could not distinguish serogroups B, D, and G, and DraI digestion could not differentiate serogroups F and I.



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  		FIG. 3. Computer-generated restriction enzyme digestion profiles of the 1-kb slpA variable region from the serogrouped strains indicated. A simulation with the four enzymes indicated is shown. Sizes of the expected restriction products are indicated in kilobases on the left.

 
Sequence analysis of the slpA gene variable region from C. difficile clinical isolates. The unique slpA sequence and RFLP patterns of each serogroup suggested that nucleotide sequence analysis and/or PCR-RFLP of the variable region of this gene could be employed to differentiate between serogroups. To test this hypothesis, we sequenced the variable region of the slpA genes from 22 clinical isolates whose serogroups were known (at least two sequences per serogroup) and which were chosen fortuitously and represented diverse geographic locations (Table 1). Analysis of the sequences obtained revealed that, with the exception of serogroup A, the nucleotide and deduced protein sequences were 100% identical within a given serogroup (Fig. 2). In contrast, the four strains belonging to serogroup A each had a unique slpA variable region, although the SlpA N-terminal regions in A1 and A10 were 93% identical.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Different methods have been developed to type C. difficile strains. A correlation has been observed between the electrophoretic patterns of proteins extracted from clinical isolates and their virulence, strains isolated during hospital outbreaks and symptomatic disease having a different profile from that observed in isolates from healthy carriers or infants (21). Delmée et al. (14) compared serogrouping of C. difficile by slide agglutination with rabbit antisera and polyacrylamide gel electrophoresis of whole-cell proteins, permitting correlation between the two typing systems and establishment of a single classification. This serogrouping is also carried out by the enzyme-linked immunosorbent assay technique.

Recently, new molecular techniques have been developed to type C. difficile strains based on DNA polymorphism, such as arbitrary primed PCR, a genotypic method permitting detection of polymorphisms within the target genome without prior knowledge of the target nucleotide sequence. This method has been used as an efficient discriminative method for investigation of nosocomial outbreaks of C. difficile-associated diarrhea but has certain drawbacks, e.g., lack of reproducibility (1, 2, 5, 7). Pulsed-field gel electrophoresis has been applied to C. difficile, but this method is costly and technically complex and some strains are untypeable (7). PCR ribotyping is being used routinely by the United Kingdom Anaerobe Reference Unit in Cardiff, Wales, to type C. difficile strains (7). This method relies on DNA pattern profiles, obtained by PCR amplification of a specific chromosomal region such as the rRNA gene (4) or the 16S-23S rRNA gene intergenic spacer region (25). Each of the strains belonging to one of Delmée's serogroups gives a different banding pattern. PCR ribotyping appears to be more discriminatory than the arbitrary primed PCR or pulsed-field gel electrophoresis method in epidemiological studies of C. difficile outbreaks (7). However, most of the aforementioned typing techniques are discriminating but labor intensive. Serotyping with specific antisera gives adequate levels of discrimination for epidemiological purposes and is not technically difficult. However, the reagents required for serotyping are not readily available to most diagnostic laboratories and some strains are untypeable or coagglutinable with this technique.

The epitopes for the serotypes are probably parts of bacterial surface proteins. In group A streptococci, sof (serum opacity factor gene) or emm (M protein gene) sequence-based analysis has been used more successfully than serological analysis for strain subtyping (3). These proteins are surface associated. Until now, no investigations have explored the usefulness of S-layer gene sequencing for subtyping of strains. A study of Campylobacter fetus (9) and Lactobacillus helveticus (32) demonstrated that amplification of the S-layer gene by PCR can be used for identification of strains. Furthermore, for C. fetus, each strain exhibited a different Southern blotting pattern when hybridized with the PCR product. This suggested that genotyping based on slpA gene structure could be useful for typing of strains. Since the P36 S-layer protein of C. difficile is located on the surface of the bacteria and because S-layer proteins are the most abundant bacterial proteins (23), it would be logical to conclude that they play a major role in determining serogroup specificity. This was confirmed by PCR-RFLP analysis and nucleotide sequencing: DNA and deduced amino acid sequences of the slpA variable region were 100% identical within a given serogroup, whereas interserogroup identity was, in general, fairly low. However, further sequencing of more strains is necessary to confirm these data. The exception is serogroup A, which is known to carry 20 subgroups. It is evident that these subserogroups may not be completely specified by the S-layer, since the slpA sequences were quite similar in serogroups A1 and A10. The subserogroup specificity of highly flagellated serogroup A could be attributable to other surface proteins, most likely the flagella.

These data indicate that the slpA gene constitutes a reliable target for differentiation of C. difficile isolates and could be used as an alternative method to serotyping, at least in an outbreak setting until more diagnostic data become available. The gene could be easily amplified from various strains by PCR by using primers described here and then sequenced. Alternatively, the amplified DNA could be digested with restriction enzymes and profiles could be compared. However, sequence-based methods have advantages over the more commonly used RFLP methods, which are difficult to standardize. Furthermore, the relatively small size of certain restriction fragments obtained after digestion may render interpretation of the profiles difficult in clinical laboratories. Sequence data give more reliable results, thus eliminating interlaboratory variation due to, for example, gel mobility differences (20). The technology for generating DNA sequence data has become readily available for on-site or commercial service companies. Finally, this methodology should have a relatively low cost; the overall cost for generating one sequence can be estimated to be less than $10 U.S. However, because of country-to-country variations in labor and reagent costs, this estimate may not be valid for some countries. Sequencing can be expected to produce the same results in different laboratories, even with the use of different methods. This, combined with the ease of generation of template DNA for sequencing by PCR, makes sequence-based molecular typing a promising alternative to the more traditional methods.


   ACKNOWLEDGMENTS
 
We thank H. Kato, Kanazawa University, Kanazawa City, Japan, for the gift of C. difficile strains GAI95600 and GAI95601.


   FOOTNOTES
 
* Corresponding author. Mailing address: Université de Paris-Sud, Faculté de Pharmacie, Département de Microbiologie, 5 rue JB Clement, 92296 ChÂtenay-Malabry Cedex, France. Phone: (33) 1 46 83 55 49. Fax: (33) 1 46 83 55 37. E-mail: anne.collignon{at}cep.u-psud.fr. Back


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 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Clinical Microbiology, July 2002, p. 2452-2458, Vol. 40, No. 7
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.7.2452-2458.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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