A total of 47 VNTR loci candidates were identified for C. difficile, and 40 were used for subsequent MLVA analysis (Table 1, Additional file 1). Initially, we found 1,526 tandem-repeat loci within C. difficile 630 using the VNTRDB software [25]. After exclusion of repeatedly detected loci, tandem-repeat loci with a copy number size >2 bp and an amplicon size of <700 bp were analyzed for variability. Finally, 47 loci exhibiting variable alleles were identified. The allelic diversity, allele number, and typing ability of all 47 VNTRs from the 142 strains were determined. Several VNTR loci with additional or imperfect repeats were observed (Additional file 1). CDR59 amplicon exhibited two adjacent VNTR loci, while CDR60, cd5, cd6, cd7, and cd25 exhibited incomplete tandem repeats. To analyze these loci in the MLVA panels, alleles of these loci were represented by repeat array size instead of copy number, and the MLVA types were analyzed with minimum spanning tree (MST) using a categorical coefficient. VNTR loci with low typing ability and/or deletions were excluded, with the CDR5, cd8, cd28, and cd20 loci amplifying at only 70%, 77%, 79%, and 79%, respectively. Additionally, deletions in amplicons from cd16, cd19, and cd39 were found. Consequently, only 40 VNTR loci were used in the following experiments.

Of the 142 isolates, capillary gel electrophoresis-based PCR-ribotyping identified 57 independent types, including 32 singletons. The most common types were R45, R4, R10, R14, and R17 (UK017), containing 7, 17, 11, 11 and 9 isolates, respectively (Figure 1). The R27 (UK 027) virulent type was not found among the local strains.

A phylogenetic dendrogram based on the PCR-ribotypes was constructed using the 142 C. difficile isolates (Figure 1). Of the 142 isolates, PCR-ribotype, MLVA34, and MLVA10, identified 57 types, 47 groups, and 45 groups, respectively. The PCR-ribotype was more discriminatory than the two MLVA groups (Figure 1). Using a threshold of >83% similarity for defining PCR-ribotype groups, all isolates were able to be divided into 47 PCR-ribotype groups, including 22 singletons. Over 87% (41/47) of the PCR-ribotype groups were specifically recognized in the MLVA34 and MLVA10 groups. However, PCR-ribotype groups 39 and 25 were recognized together as one by both MLVA groups, with the fingerprints for these isolates sharing a 70% similarity (a four-band difference). In addition, PCR ribotype groups 26 and 49 were also identified as one by the two MLVA groups, with the fingerprints of these two isolates sharing a 78% similarity. Furthermore, PCR ribotype groups 8 and 23 were also seen as one by the two MLVA groups, with the fingerprint of these isolates sharing an 82% similarity. Taken together, these results shows that this discordance, the lack of one to one identification between PCR ribotypes and MLVA groups, mainly occurred when PCR-ribotypes shared >83% similarity.

MLVA panels with slightly limit allelic diversity generated groups highly congruent with PCR ribotyping (Table 2). To determine the most congruent groupings between MLVA panels and PCR-ribotype groups, groupings of MLVA panels consisting of VNTR loci with high to low allelic diversity were compared with the PCR-ribotype groups. MLVA34, MLVA12, and MLVA10 generated partitions (47, 45, and 45, respectively) and allelic diversity (0.959, 0.957, and 0.957, respectively) similar to those identified by PCR ribotyping (Table 2). The congruence of the grouping of MLVA34, MLVA12, and MLVA10 with the PCR ribotype groups amounted to 97.3, 98.9, and 98.9%, respectively (Table 2). These values were significantly higher than that of the MLVA40 group (2.6%). MLVA40, which included six highly variable VNTR loci, C6cd, CDR60, CDR4, CDR49, CDR9, and CDR48 (allelic diversity: 0.84-0.96), generated a lot more partitions (136) and higher allelic diversity (0.999) than PCR ribotyping. In most PCR-ribotypes, multiple alleles were observed for C6cd, CDR60, CDR4, CDR49, CDR9, and CDR48 loci (Additional file 2), whereas the other 34 VNTR loci exhibited little variance. This data indicates that the greatest discrepancy between groupings in these two methods occurred in loci with high allelic diversity, and that congruence increased when the highly-allelic-diversity loci were removed, as in MLVA34.

To identify a simplified panel resembling MLVA34, the groups from three smaller panels (MLVA12, MLVA10, and MLVA8) were evaluated for agreement with the PCR-ribotype groups. MLVA10 was the simplest panel yielding groups that were highly congruent (98%) with the PCR-ribotype groups (Table 2). In contrast, congruence significantly decreased when the MLVA was simplified to just eight VNTR loci.

MST analysis revealed that the MLVA34 types could be clustered into 47 groups, including 21 singletons (Figure 2). Most (41/47) of the MLVA34 groups were specifically recognized as a single PCR-ribotype group, except for 34_4, 34_41, 34_11, 34_48, 34_25, and 34_26. An isolate of the group 34_41 could not be typed by the cd7 and cd34 loci, and was separated from those of the 34_4 MLVA group; however, all isolates of the 34_41 and 34_4 groups belonged to PCR-ribotype group 4. This shows that isolates of the 34_4 and 34_41 groups were closely related. Isolates of group 34_11 and 34_48 were separated by their different allele numbers at CDR59 and H9cd loci, but these two MLVA groups both belonged to the PCR-ribotype group 11.

MST analysis revealed that the MLVA10 types could be clustered into 45 groups, including 20 singletons (Figure 3), and most (41/45) of the MLVA10 groups were specifically recognized as a single PCR-ribotype group. The clustering of MLVA10 (Figure 3) yielded groupings similar to those of MLVA34, except for isolates of PCR-ribotype groups 4, 8, and 23. Since the cd34 VNTR locus was not used in the MLVA10 panel, isolates from the PCR-ribotype group 4 all belonged to the 10_4 group. This indicates that the MLVA10 panel was able to type more strains than the MLVA34 panel. In addition, isolates of the PCR-ribotype groups 8 and 23 were grouped into the 10_8 group, indicating that the MLVA10 is less discriminatory than MLVA34.

MLVA panels containing different numbers of VNTR loci were used for discriminating 142 C. difficile isolates into different genotypes and the Simpson's index of diversity (ID) was shown to increase with the number of VNTR loci used (up to MLVA4; Table 3). Using MLVA4, 142 isolates were grouped into the largest partitions (140). MLVA4 was shown to be as discriminatory as MLVA40 using all 40 VNTR loci (Table 3). However, when the MLVA panels contained fewer than three VNTR loci, the partitions decreased significantly.

MLVA4 and MLVA10 were used for classifying 59 isolates acquired from a hospital in central Taiwan, and four clusters were identified (Figure 4; Additional file 3). These clusters consisted of three independent clusters (B, C, and D) containing two isolates each from inpatients and one (A) cluster containing two isolates from outpatients during the ten month surveillance. Each of the two isolates from the B, C, and D clusters were recovered from different pediatric patients with 3, 0, and 4-days intervals of specimen submission by the physician from children's ward, respectively (Additional file 3). The two isolates from cluster A were shown to differ at one locus (1/14) in the combined MLVA4 plus MLVA10 panel and were isolated from two specimens of the same patient within a four-day interval. Most isolates were non-toxigenic strains, except those in cluster D. The patient in the D cluster developed diarrhea and was infected with toxigenic C. difficile strains that were assigned to C. difficile infection cases. On the other hand, a single PCR-ribotype group was usually grouped with less than five VNTR loci differences (5/14).

A total of 142 C. difficile strains that were either toxigenic or non-toxigenic were used in this study. Five reference strains (NCTC11204, NCTC13366, NCTC13287, NCTC13404, and NCTC13307) were purchased from the National Collection of Type Cultures (NCTC, London, UK) and three reference strains (BCRC17900, BCRC17702, and BCRC17678) were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). One strain (NAP1/027) was kindly provided by Dr. Brandi Limbago from the United States Centers for Disease Control and Prevention (CDC), and 133 strains were isolated from clinical laboratory specimens in Taiwan. Among local isolates, 73 strains were isolated from outpatients, and 60 strains were isolated from hospitalized patients that were comprised of 38 from adult wards and 22 from children's wards.

All specimen strains were provided by five clinical laboratories between November 27, 2007 and December 31, 2008. The corresponding epidemiological data for each strain were provided by clinical laboratory staff. Four laboratories were located in central Taiwan, and one laboratory in the southern part of Taiwan. All five clinical laboratories cultured all available stool or rectal-swab specimens on Cycloserine Cefoxitin Fructose Agar (Oxoid, Hampshire, UK) and the plates were incubated under anaerobic conditions for 48 h. All suspected C. difficile colonies were sent in an anaerobic pack and delivered within 24 h to the central-region laboratory at the Centers for Disease Control in Taiwan for further identification. All purified isolates were stored in 15% glycerol at -80°C.

All suspected C. difficile colonies were analyzed for a species-specific internal fragment of the triose phosphate isomerase (tpi) housekeeping gene, and toxigenic type was characterized by PCR amplification of internal fragments of the toxin A gene (tcdA) and the toxin B (tcdB) gene, as previously described [39]. Briefly, each candidate colony was dissolved in 1 mL distilled water and then boiled for 15 min to prepare DNA. Tpi-, tcdA-, and tcdB-specific primers [39] were used in independent PCR reactions. PCR was performed in 20 μL volumes containing the following components: 50 ng DNA, 10% glycerol, 0.5 μM of each primer, 200 μM dNTPs, and 1 U of Taq DNA polymerase (BioVan, Taiwan) in a 1× amplification buffer solution (10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl₂). PCR was performed on a GeneAmp System 2400 thermal cycler (Applied Biosystems). The PCR cycle conditions were as follows: 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 3 min. PCR products were resolved by electrophoresis on a 1.5% agarose gel stained with ethidium bromide.

The full-length sequences of C. difficile QCD-32g58 and C. difficile 630 were compared using VNTRDB software [25] to find tandem repeat loci in the genome. Tandem repeats with a repeat length >2 bp and ≥70% consensus match were initially selected for screening by PCR from the BCRC17678 and BCRC17702 reference strains and four experimental isolates. Primers that flanked the tandem repeat region were designed using the online Primer 3 software (http://frodo.wi.mit.edu/primer3; Additional file 5). VNTR screening was initially performed by PCR amplification of each candidate tandem repeat locus in genomic DNA from six isolates. The variability of each tandem repeat locus was assessed by gel electrophoresis on a 1.5% agarose gel, and sequence analysis was performed to determine the size of the resulting PCR products and the tandem repeat copy number.

To find a MLVA panel most congruent to PCR ribotyping, 40 VNTR loci were sorted by allelic diversity and then arranged to form various panels by sequentially removing the highest allelic diversity loci. Each panel was compared with PCR ribotyping, and the congruence between the two techniques was calculated using the Rand coefficient [40].

The simplest MLVA panel that would yield a MLVA34-like genotype distribution of 142 C. difficile strains was found as follows. First, the partitions given by each of the 34 VNTR loci were calculated based on Wallace coefficients to evaluate their predictable value by the other 33 loci. Loci that showed either more predictability or lower allelic diversity than other loci in the MLVA34 panel were excluded. There were 22, 24, and 26 loci excluded when the predictable values were higher than 75, 70, and 65%, respectively. This exclusion resulted in the MLVA12, MLVA10, and MLVA8 panels (Additional file 6). All MLVA panels were analyzed by the minimum spanning tree (MST) method, and the concordance between MLVA groupings and PCR-ribotype data were calculated.

Genomic C. difficile DNA was purified using the QIAamp DNA Mini kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions. Genomic DNA isolated from C. difficile were then used for PCR amplification of VNTR and PCR ribotyping.

PCR amplification of the 47 VNTR candidates was performed on six strains with the primer sets shown in Table 1. Each PCR was performed in a 10 μL reaction containing the following reagents: 25 ng genomic DNA, 1 μL buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl₂; BioVan, Taiwan), 250 μM MgCl₂, 1% DMSO (Sigma-Aldrich, St. Louis, MO), 200 μM dNTPs, 0.5 μM primer set, and 1 U Taq DNA polymerase (BioVan, Taiwan). The PCR cycle conditions were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 40 s, 50°C or 52°C for 90 s, and 72°C for 50 s, and a final extension at 72°C for 3 min. Sequence analysis of the PCR products was performed by Mission Biotech Corporation with the ABI Big Dye Terminator Kit v.3.1 (Applied Biosystems) and the same primers used for PCR.

PCR amplification of the 48 selected C. difficile VNTR loci was performed on DNA extracted from 142 C. difficile isolates. The primer sets, annealing temperatures, and primer panels are shown in Additional file 5. Amplification of the 47 VNTR loci was carried out in 12 multiplex PCR reactions and one single PCR reaction (Additional file 5: M1-M13). Amplification of the 14 VNTR loci of MLVA4 and MLVA10 was carried out in four multiplex PCR reactions (Additional file 5: M14-M17). The PCRs were performed in 10 μL reactions containing the following reagents: 25 ng genomic DNA, 1 μL buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl₂; BioVan, Taiwan), 250 μM MgCl₂, 1%DMSO (Sigma-Aldrich, St. Louis, MO), 200 μM dNTPs, 0.02-0.1 μM primer set, and 1 U Taq DNA polymerase (BioVan, Taiwan). The PCR cycle conditions were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 40 s, annealing temperature for 90 s, and 72°C for 50 s, and a final extension at 72°C for 3 min. Fragment analysis of the multiplex PCR products was performed as follows: 1 μL of each 20-fold-diluted PCR product, 0.1 μL GeneScan 500 LIZ size standard (Applied Biosystems, Warrington, UK) and 8.9 μL HiDi (Applied Biosystems, Foster, CA) were mixed and denatured at 95°C for 5 min. The products were then analyzed on an ABI3130 sequence detection system (Applied Biosystems). The obtained fragment sizes were exported as an Excel spreadsheet file (Microsoft, Redmond, WA). The corresponding copy numbers were calculated by comparison to the size of reference strains using Excel software (Microsoft). The equation used for calculation of copy number is as follows:

Copy number of VNTRn = [(Fs-Fr)/repeat size of VNTRn] + copy number of reference strain, where Fs, fragment size of test strains in each VNTR loci; Fr, fragment size of reference in each VNTR loci; VNTRn, either locus in 40 VNTR loci.

Genomic DNA from all the C. difficile strains was amplified with the primer set designed by Bidet et al. [18], and the electrophoresis-based PCR-ribotyping was performed using a method modified from Indra et al. [19]. Briefly, the primer was labeled with carboxyfluorescein (FAM) dye to enable DNA sequence analysis. The PCR mixture included the following reagents: 25 ng genomic DNA, 1 μL buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, and 1.5 mM MgCl₂; BioVan, Taiwan), 200 μM dNTPs, 1.5 mM MgCl₂, and 1 U Taq polymerase (BioVan, Taiwan) in a 20 μL final volume. One microliter of each 20-fold-diluted PCR product, 0.8 μL Genflo625 ROX-labelled DNA Ladder (Chimerx, USA), and 8.2 μL HiDi (Applied Biosystems, Foster, CA) were mixed and denatured at 95°C for 5 min and then analyzed with a ABI3130 sequence detection system. The ribotype fragments for the full-length sequencing of strain NCTC13307 (C. difficile 630) were first predicted by the PCR-amplification function from in silico analysis using the website (http://insilico.ehu.es), and the curve file from the ABI sequencer was confirmed by the predicted size. Ribotypes 001, 012, 017, 027, and 106 were set up by comparing the curve files with the five reference strains NCTC11204, NCTC13307, NCTC13366, NCTC 13287, and NCTC13404, respectively. All PCR-ribotypes were named with an "R" prefix before the serial number.

The allelic diversity of each VNTR locus was measured by its Simpson's index [41] and confidence interval (CI) [42]. The ability of each VNTR locus to type the 142 isolates was measured as follows:

Number of isolates amplified in each VNTR locus/142.

The copy numbers of the VNTR loci from all of the 142 isolates were imported into the Bionumerics software (Applied Maths, Belgium), and the categorical coefficient and the highest number of single-locus-changes were used for the minimum spanning tree construction [43]. The curve files of all the ribotypes from the ABI sequencer were imported into the Bionumerics software for further standardization. The PCR-ribotyping fingerprints of all the isolates were analyzed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering algorithm, using the Dice coefficient (tolerance: 0.2%). The quantitative level of congruence between the typing techniques was based on the adjusted Rand (AR); the predictable value between VNTR loci was based on Wallace's coefficients, using an online tool for the quantitative assessment of classification agreement (http://darwin.phyloviz.net/ComparingPartitions) [40].