Clostridium difficile is the most commonly recognized cause of infectious nosocomial diarrhea [1]. Illnesses associated with C. difficile range from mild diarrhea to pseudomembranous colitis and toxic megacolon [2]. In the early 2000s, an emerging virulent strain, NAP1/027, caused hospital outbreaks in Canada [3], and later, strains of the same genotype were also found in the United States of America, Europe, and Asia [3–5]. To understand the spread of bacteria and identify clones with apparent increased virulence, several molecular methods for genotyping have been used to investigate C. difficile [6–10]. Multilocus sequence typing (MLST) is the "gold standard" for assessing population structure. Polymerase chain reaction (PCR) ribotyping has been used for the global analysis of related virulent strains based on a reference library involving 116 genotypes acquired since 1999, and has become the most common technique to represent the epidemic clone of C. difficile [11]. In addition, pulsed-field gel electrophoresis (PFGE), surface layer protein A gene-sequence typing (slpAST), restriction endonuclease analysis (REA), and multilocus variable-number tandem-repeat analysis (MLVA) have been used for outbreak studies of C. difficile [7, 8, 12–14]. Among these techniques, MLVA panels exhibit a significantly higher discriminatory power (allelic diversity: 0.964) than PFGE, slpAST, and PCR ribotyping [9]. As a result, MLVA has been the most commonly used to distinguish strains from different outbreaks, whereas PCR ribotyping and PFGE have mostly been used to detect long-term relationships among strains when compare to MLVA [15, 16].

PCR ribotyping is performed using a PCR-based method to detect polymorphic sequences in the 16S-23S intergenic spacer region (ISR) in C. difficile [17]. The band-pattern data generated by this method is difficult to transport and to compare between laboratories [18, 19]. Therefore, a few studies have tried to replace PCR ribotyping with other methods [19–22]. Typing of slpA, which is based on the S-layer gene sequence of C. difficile, recognizes only nine of the 14 PCR-ribotypes [22]. Recently, a highly discriminatory MLST method based on seven housekeeping genes (adk, atpA, dxr, glyA, recA, sodA, and tpi) sequences was develop to allow genotyping of C. difficile; the resulting sequence type (ST) recognized 32 of 40 PCR-ribotypes [21]. To date, the tandem repeat sequences type (TRST) technique is the most concordant method; this method, which combines two variable tandem repeat sequences, resolved the phylogenic diversity at a level equivalent to PCR ribotyping [20]. The MLVA employs multiple variable-number tandem-repeat (VNTR) loci with varying levels of diversity to resolve genetic relationships. VNTRs with a high degree of diversity are used to differentiate closely related strains. In addition, recent research in Staphylococcus aureus and Neisseria meningitidis showed that VNTR loci with a lower degree of diversity can establish deeper phylogenetic relationships consistent with the MLST method, which is based on the slowly-mutating housekeeping gene sequences [23, 24]. In the past, for C. difficile, the MLVA panel has been found a more discriminatory method than PCR-ribotyping [13, 14]. In this study, we hypothesize that an MLVA panel with a lower combined allelic diversity may be more congruent to PCR ribotyping.

The purpose here was to determine a MLVA panel that could yield results in accordance with PCR ribotyping results. Serial MLVA panels were compared with PCR-ribotype groups based on an investigation of 142 C. difficile isolates. By combining more conserved VNTR loci, we found MLVA10 had excellent congruence with the epidemic clone. Moreover, a simple MLVA (MLVA4) with high discriminatory power was also proposed as a useful alternative. Therefore, MLVA10 and MLVA4 can be combined in four multiplex PCR reactions to save operation time when typing a large collection of isolates.

A MLVA system is composed of VNTR loci that exhibit varying levels of diversity, and can be employed either for long-term or short-term investigations [26]. In the present study, we proposed two MLVA panels, MLVA10 and MLVA4, for the differentiation of C. difficile isolates. MLVA10 exhibited a slightly lower allelic diversity than previously identified panels [13, 14], and is recommended as a complementary test to the PCR-ribotype groups. MLVA4, in contrast, exhibited high allelic diversity and is recommended for the detection of short-term evolution in strains of C. difficile.

In the current study, except for nine reference strains, the 133 local isolates were a widely distributed collection and none were previously reported as outbreak strains by clinical laboratories. These isolates were acquired from patients 0.1-88 years of age and contained 73 isolates from outpatients that were assumed to be community-acquired strains. The other 60 isolates were recovered from hospitalized patients, with 38 collected from children's wards and 22 from adult wards. In addition, this study involved 57 PCR-ribotypes (Table 3), a considerably higher number than previously reported [9]. Therefore, the sample population used in the current study is proposed to be more suitable for comparison between the two methods [20, 21, 27]. In the ribotype distribution, it is noteworthy that the PCR-ribotype R17 (UK 017), a clone found worldwide and is related to an animal source (in addition to 027 and 078 types) was the fourth (9 in 142) most frequently identified type in this study (Figure 1) [28, 29]. In the current study, the R17 type was only found in samples obtained from central Taiwan, but the exact distribution of PCR-ribotypes requires further investigation using a more precise sampling method. Furthermore, PCR-ribotypes other than 001, 017, 027, and 106 should be compared with standard PCR-ribotypes from the European reference laboratory.

While comparing PCR ribotyping to other techniques, allelic diversity was identified as an important factor. Previous studies identified that slpA type did not have high enough variability to differentiate all PCR-ribotypes [22]. The current study found that the CDR4, CDR9, CDR48, CDR49, CDR60, and C6cd VNTR loci [13, 14, 19] used in previous MLVA panels were variable in each PCR-ribotypes (Additional file 2); this made these panels too discriminatory for congruency with the PCR-ribotypes here. In contrast, the highly discriminatory MLST method had an index of discrimination of 0.9, similar to that of the PCR-ribotype (0.92), and the resulting ST recognized 80% of the PCR-ribotypes [21]; the TRST resulted in an allelic diversity (0.967) equal to that of PCR ribotyping (0.967), and is the technique most related to PCR ribotyping among these studies [20]. In the present study, the ten VNTR loci used in MLVA10 were cd5, cd6, cd7, cd12, cd22, cd27, cd31, H9cd, F3cd, and CDR59, which exhibited a slightly lower allelic diversity (0.54-0.83) than the previously used CDR4, CDR9, CDR48, CDR49, CDR60, and C6cd VNTR loci (0.84-0.96) [13, 14, 19, 20] (Table 1), resulting in a combined allelic diversity of 0.957 (Table 2). This value is similar to TRST (0.967) and PCR-ribotype (0.967). Therefore, both TRST and MLVA10 showed a high level of agreement with the PCR-ribotype (86.0 and 88.2%, respectively) (Table 2). However, the MLVA technique is easier to perform than the sequence-based techniques, such as TRST and MLST, and MLVA panels are more easily combined, such as when adding the MLVA4 panel for outbreak strain detection.

To represent the currently known PCR-ribotypes for C. difficile, a combination of multiple VNTR loci with different allelic diversity is recommended. In our initial study, no single VNTR locus was discriminatory enough to recognize all PCR-ribotypes or specific enough to belong to each PCR-ribotype (data not shown), as previously observed for MLVA and MLST of N. meningitidis [24]. Therefore, 40 VNTR loci distributed throughout the genome of the C. difficile 630 strain were used for comparison analyses, and we found that the MLVA34 panel yielded groups most related to the PCR-ribotype groups (Table 2; Figure 1). Our screening method was based on two rationales: 1) the PCR-ribotype recognized the major PFGE type [9] and was expected to be congruent with the major genotypic groups of C. difficile; and 2) the locus markers distributed throughout the chromosome were more likely to identify genotypic change [13].

In the current study we also highlighted the fact that group definition was required for comparisons. The allelic diversity of MLVA10 types varied among the different PCR-ribotypes (Additional file 4), and led to only 60% congruence between the types of MLVA10 and PCR ribotyping (data not shown). In significant contrast, the congruence reached 98% when groups obtained by the two techniques were compared (Table 2). These observations were similar to those found in the comparison between MLVA34 and PCR-ribotyping (Additional file 4). Even though there was a high level of agreement between groups identified by the two techniques, some discordance was found. For example, PCR-ribotype group 11 was represented by two MLVA10 groups (10_48 and 10_11) (Figure 1), and the isolates in group 11 were suspected to have undergone concerted evolution [30, 31]; however, this assumption needs to be further confirmed by MLST.

For the detection of outbreak strains, two MLVA panels, each composed of seven VNTR loci, have been developed. One panel consisted of CDR4, CDR5, CDR9, CDR48, CDR49, CDR59, and CDR60, and the other panel consisted of C6cd, H9cd, F3cd, CDR4, CDR9, CDR48, and CDR49 [13, 14]. However, our study indicated that MLVA4, which consisted of C6cd, CDR4, CDR49, and CDR60, was able to discriminate all 142 test strains (Table 3), as previously observed for MLVA of Salmonella typhimurium [32]. Furthermore, all of these VNTR loci exhibited higher allelic number and copy number variation than previously reported (Table 1) [14]. Our results may be explained by two reasons: 1) among these loci, CDR60 loci was found exhibit incomplete copy number and was assigned by repeat array size, as this could increase the allelic number; and 2) we validated these loci in a more random population than previous studies [13, 14], which would increase the value of allelic diversity. In addition, we used a categorical coefficient instead of STRD to analyze the MLVA data and to analyze the loci represented by the repeat array size. Although this may reduce the sensitivity to differentiate the outbreak strains, analyses using the STRD coefficient were found to be too variable and may obscure the epidemiological links between C. difficile outbreak strains when several repeats at a locus are deleted or duplicated simultaneously [33].

All clusters detected by MLVA4 and MLVA10 combined can be explained by epidemiological information. Apart from the two patients from cluster D were C. difficile infection cases, other patients from other clusters were assumed to be C. difficile carriers (Figure 4; Additional file 3). The major limitation of this validation for the study of outbreak strains was the sample population we used; the 142 test strains used in the current study were a randomly sampled population that did not contain outbreak strains, and the genetic relationship between these was distant. For these reasons, this may have overestimated the discriminatory power of the MLVA 4. Therefore, the MLVA4 panel requires further validation using closely related strains, such as outbreak strains from hospitals, before any conclusions as to its discriminatory power can be made.

Five imperfect VNTR loci (cd5, cd6, cd7, CDR59, and CDR60) were used in this study, except for CDR59, the other four loci were long-repeat VNTR loci with incomplete repeats (Additional file 1). The incomplete repeats may be caused by insertions and deletions, which often result in horizontal gene transfer between bacteria strains and obscured the phylogenic relationship in the bacteria population [34]. However, the long-repeat regions exhibited a higher frequency of recombinations, and were considered attractive candidate regions that could be used for determining phylogenetic relatedness between species and strains [35]. The long-repeat VNTR loci have been known to be responsible for adaptive evolution, as for antigenic variation [34], and were also used to differentiate the C. botulinum and N. meningitides[36, 37]. Therefore, we analyzed these imperfect VNTR loci for use in the screening for appropriate panels that showed agreement PCR-ribotyping. Our data showed that cd5, cd6, and cd7 loci did not decrease the congruency with PCR-ribotyping (Table 2; Additional File 2). The result may be due to that the 16S-23S intergenic spacer region, on which the PCR-ribotyping based on, was not as conserved as a housekeeping gene that is used to construct the phylogenic tree [9, 38]. However, the variations from these incomplete repeat loci should be detected in our follow-up surveillance.

PCR ribotyping is a standard technique used worldwide for epidemic clone detection, but the ambiguous data generated by this technique is difficult for assessing inter-laboratory efficacy. MLVA is a fast and easy-to-use method, and its numerical profile output is more transferable than the standard PCR ribotyping technique. In our laboratory setting, the cost of PCR ribotyping, MLVA10, and TRST per isolate was $0.87, $2.53, and $13.60, respectively, and the cost of the most recent MLST is $24.65 according to Griffiths' estimation [21]. In the current study, the cost of MLVA10 was slightly higher than that of PCR ribotyping, but was still significantly less expensive than the TRST and MLST sequence-based typing techniques. Moreover, when analyzing a large number of isolates, it is simpler to perform one genotyping technique than multiple techniques. Taken together, the MLVA10 is recommended for the detection of C. difficile PCR-ribotype groups and for use in combination with the MLVA panel designed for the detection of outbreak strains. Future studies will involve evaluation of MLVA10 for its phylogenetic information by comparison to MLST typing.

For the classification of C. difficile strains, the MLVA technique can result in a distinguishable data set that is more useful for comparison and is highly congruent with PCR-ribotype results. The MLVA10 panel may be used either to detect the PCR-ribotype groups or to overcome the drawbacks of the PCR ribotyping technique. In addition, the MLVA4 can be used to detect closely-related strains. These two MLVA panels can be combined and used for epidemiological studies of C. difficile.