strain subtyping is widely practiced in the context of human listeriosis surveillance and food safety control. Because the typing methods that are currently widely used, including serotyping, PFGE, and MLST, present a series of limitations, MLVA has attracted intense interest. MLVA fulfills a number of criteria recognized as important for the successful implementation of a typing method and the interpretation of its results (46
). In particular, the rapidity, low cost, and easy implementation of MLVA are practical advantages that are largely recognized (23
). In the present study, we have developed an optimized MLVA scheme as a typing method for L. monocytogenes
and compared it to two reference methods, MLST and PFGE. We have exhaustively tested the previously published VNTR primer sets and defined new ones against a genetically diverse set of L. monocytogenes
strains. This allowed us to (i) develop a simple MLVA typing system, MLVA-11, which is based on two multiplex PCR assays combining 11 selected VNTR loci; (ii) analyze the variation at these 11 loci among a collection of 255 isolates; and (iii) compare the MLVA data obtained with PFGE and MLST data.
Previous MLVA schemes have been associated with a number of PCR failures for some VNTR loci, resulting in the incomplete characterization of some isolates (26
). Missing data lead to lower discrimination and loss of informative characters for phylogenetic placement and render the interpretation of differences among MLVA profiles problematic. Here, we optimized the selection of VNTR loci and the sequences of primers. We thus achieved nearly complete (99.9%) typeability for 10 markers, even when combining these assays into two multiplexed PCRs. As previously reported (29
), we found null alleles at locus Lm11, but these PCR failures were restricted to isolates of CC7 and CC8, consistent with the absence of the corresponding locus in the genomes of representative strains of these two clones. However, we chose to keep this locus in the M1 multiplex assay because it provides some discrimination within CC1, one of the epidemiologically most important clones of L. monocytogenes
. Besides, the inclusion of the Lm11 primers in the M1 multiplex PCR mixture did not negatively affect the assay for the other loci. As our sample covers a large breadth of the clonal diversity of lineages I and II, including all of the clinically most frequent serotypes and clonal groups, we demonstrate that the MLVA-11 system has broad applicability, an important characteristic of typing systems (46
While this work was in progress, two studies were published in which distinct MLVA schemes were compared and combined (31
). Our MLVA scheme includes four loci that were not used by Li et al., while these authors' scheme has two loci that we chose not to include (LM-TR3 and LM-TR6). Chen et al. combined the three VNTR loci from Murphy et al. (28
) with the three loci from Miya et al. (27
). However, LM4b-TR1 (27
) and LM-TR3 (28
) correspond to the same locus and are therefore redundant. Besides, locus LM-TR3 was removed from our scheme as variation at this locus is difficult to interpret because of size variation in the regions flanking the repeat array (29
Allele sequencing allowed us to confirm that size variation at the 11 selected loci is attributable to repeat number differences. We showed that MLVA loci are stable during laboratory subculture and found complete repeatability of allele coding by capillary electrophoresis separation. Elimination of loci with nonspecific amplification also facilitated the sizing of fragments and the determination of tandem repeat numbers. We also showed that the L. monocytogenes VNTR loci selected have a very strong tendency to evolve by the stepwise addition or removal of single repeat units. This observation indicates that the quantitative difference in the number of repeats, rather than simple allelic mismatch, can be taken into account to estimate strain relationships.
We compared MLVA-11 with the three widely used methods serotyping, PFGE, and MLST. While several MLST schemes have been published (9
), only the Institut Pasteur scheme (12
) provides a standardized nomenclature through a publicly accessible database and is used in a coordinated manner by multiple laboratories (www.pasteur.fr/mlst
). With this MLST scheme, we characterized for the first time reference strains of several past outbreaks and show that previously defined ECs appear to correspond to MLST-defined major clones. Our MLVA assay successfully discriminated all MLST CCs, including those that correspond to ECI, ECII, ECIII, and ECIV. MLVA-11 thus appears as a very powerful method to identify these clones, as MLVA patterns are clone specific and as cluster analysis of MLVA patterns is strongly concordant with MLST clones. This good agreement can be explained by the fact that MLVA and MLST markers have similar levels of variation and by the rarity of recombination among L. monocytogenes
strains. MLVA could therefore be used as a rapid identification tool for epidemiologically important clonal groups. For this purpose, it will be important to elaborate a more complete MLVA-MLST dictionary by mapping every novel MLVA pattern onto the MLST diversity. Along the same lines, one previous study compared MLVA with MLST data and found that MLVA could be useful for the recognition of three ECs of serotype 4b (27
). It is interesting that the two multiplex assays of the MLVA-11 system may be used for different purposes. Whereas M2 multiplex alone clustered strains according to their clone and could therefore represent a useful rapid clone identification method by itself, in turn, M1 PCR was more useful to discriminate among isolates within clones.
One advantage of molecular typing methods, in contrast to serotyping, is that genetic markers may be used to estimate phylogenetic relationships among strains. MLVA is generally regarded as an unreliable phylogenetic method because of the high frequency of homoplasy. Such events can correspond either to reversion to ancestral states or to convergent evolution leading to the same allele by independent changes in different lineages (48
). We calculated that approximately half of the changes in our data set were evolutionary reversions or convergences. Therefore, MLVA data comprise a substantial degree of phylogenetic noise and must be interpreted with caution. As most VNTR loci are located within genes putatively encoding surface-exposed proteins (), it is possible that the number of repeats is subjected to selective pressures. In this context, it is remarkable that cluster analysis of the MLVA-11 profiles did recover the two main subdivisions corresponding to the two major phylogenetic lineages, I and II, and classified strains according to their MLST clone (). This result indicates that despite homoplasy, MLVA variation in L. monocytogenes
does convey useful phylogenetic information. Several MLVA alleles are largely conserved within either lineage I or II; for example, Lis-TR1317 has predominantly allele 4 in lineage I and allele 3 in lineage II, while locus JLR4 has mostly alleles 8 and 4 in lineages I and II, respectively. This remarkable stability within lineages indicates that some VNTR markers diversify very slowly in L. monocytogenes
. Another requirement for MLVA to convey phylogenetic information is that these loci must undergo restricted amounts of genetic recombination among L. monocytogenes
strains, which is consistent with low recombination rates estimated on the basis of MLST and full-genome sequence analyses (12
). It is also important to use a relatively high number of VNTR markers, as the use of each multiplex individually did not recover phylogenetic placements as accurately (data not shown).
Listeriosis is a global public health issue that led to the implementation of surveillance systems in several countries in the European Union, the United States, and Canada (18
). In this context, the high discriminatory power of PFGE made this method the de facto gold standard typing method. Our study indicates that MLVA has lower discriminatory power than PFGE based on enzymes ApaI and AscI, which is the current standard. Two previous evaluations of MLVA have concluded that MLVA had better discrimination than PFGE (26
). However, PFGE in these studies was performed with a single enzyme, either ApaI or AscI, and were thus based on a less discriminatory implementation of PFGE. Sperry et al. (29
) compared MLVA data with PFGE data for 123 isolates and demonstrated a lower discrimination of MLVA than ApaI and AscI PFGE, a conclusion with which our findings fully agree. Our scheme includes seven of the eight loci included in the scheme of Sperry et al. and four additional loci. Surprisingly high discrimination was achieved by Li et al. (33
) on the basis of nine MLVA loci, possibly because of the inclusion of solely unrelated strains, many of which were from food and the environment. From the above, we conclude that MLVA cannot be viewed as a replacement for PFGE when discrimination is a key requirement.
Interestingly, we showed that the discrimination of MLVA, relative to that of PFGE, is highly dependent on the clone. In particular, MLVA should be a useful addition to PFGE for the discrimination of strains that belong to CC4 or CC9. It is conceivable that MLVA loci evolve faster in some clonal lines, even though the reasons behind this heterogeneity are currently unknown. Alternatively, mobile elements including insertion sequences and phages may be less dynamic in some clones.
The high number of distinct MLST STs per clone and the Simpson index of discrimination of the MLST method () should not be taken as evidence that MLST has higher discrimination power than MLVA. Indeed, strains with distinct STs were purposely included in this study on the basis of our previous MLST analyses (12
) in order to test the ability of MLVA to identify all of the variants within clones. This selection has inflated MLST's Simpson index artificially. However, as multiple isolates with the same ST or closely related ones were not discriminated by MLVA, it indicates that MLVA does not subtype MLST clones efficiently, with the exceptions of CC4 and CC9.
Concordance of typing results with epidemiological information is a desirable characteristic of a typing method (46
). In our study, we used mostly independent isolates collected over wide temporal and geographic scales. Nevertheless, we found several groups of epidemiologically unrelated isolates with the same MLVA pattern and sometimes also the same PFGE pattern (see Table S1 in the supplemental material; PFGE16/54 and MLVA0010 [n
= 9], PFGE27/11 and MLVA0055 [n
= 6], PFGE53/14 and MLVA0048 [n
= 7], and PFGE42/41 and MLVA0022 [n
= 5]). These results show that identity of MLVA and PFGE profiles does not necessarily imply a direct epidemiological link. This is especially true for MLVA, and in our retrospective study of isolates from the French pork jelly outbreak, some of the isolates defined as unrelated by PFGE were not distinguished by MLVA.
MLVA has strong potential for interlaboratory standardization, as this method is highly reproducible and as data scoring into integer numerals provides unambiguous results. This is largely regarded as an advantage over PFGE, which can imply partly subjective decisions during band scoring. Standardization would provide benefits to international surveillance and population biology. However, MLVA standardization requires calibration of fragment sizing apparatuses and interlaboratory reproducibility needs to be carefully evaluated. For this purpose, we identified a set of 12 isolates that display distinct alleles at each of the 11 loci and together represent 80% of the distinct alleles found in this study (see Table S1 in the supplemental material). This MLVA reference set of strains is available upon request and should constitute a useful resource for interlaboratory calibrations.
MLVA was implemented in this study in the form of two multiplexed PCRs combining a total of 11 VNTR markers. The variation disclosed at these loci proved highly consistent with MLST data and was phylogenetically informative. These results show that MLVA could be used as a rapid identification method for MLST-defined clonal groups, including those corresponding to so-called ECI to ECIV. Because it has lower discriminatory power, MLVA cannot replace PFGE in outbreak investigations. However, given its simplicity, low cost, high throughput, and rapid time to results (around 8 h), MLVA could represent a useful screening method to alleviate the PFGE workload. Within the context of an outbreak, MLVA could advantageously fill the gap between the throughput needed to characterize a high number of isolates in a short period of time and the high discrimination level needed for informed epidemiological decisions. MLVA may also represent a suitable first-line assay for listeriosis surveillance, with PFGE efforts being focused on common MLVA genotypes. A two-step MLVA-PFGE strategy could significantly lighten the workload and would position MLVA as an important new tool in listeriosis surveillance.