Within the Y. pestis species, strains are separated into three biovars according to their ability to reduce nitrate and to ferment glycerol 1. Since Y. pestis was connected to plague by Yersin 2, strains of biovar Antiqua have been generally isolated from Asia and from East and Central Africa, Medievalis was found in Central Asia, and Orientalis worldwide. Y. pestis is thought to have recently evolved from Yersinia pseudotuberculosis, some 1,500 to 20,000 years ago 3. Based on the biochemical assays, Devignat 1 proposed that Antiqua strains, causing probably the first known pandemic, represent the ancestor. Medievalis strains are suggested to be responsible for the second pandemic whereas the third pandemic was associated exclusively with Orientalis strains. This overall scenario, although not formally established, is supported by the observed higher diversity in the Antiqua and Medievalis biovars as measured by IS typing and the geographic origin of strains 345. "Pestoides" strains are particular Y. pestis isolates found recently in Russia, and which infect unique species of rodents 56.

Several molecular methods have been used for genotyping Y. pestis strains, mostly based on pulse-field gel electrophoresis (PFGE), insertion sequence polymorphism and ribotyping. Recently, multiple locus variable-number-of-tandem-repeat (VNTR) analysis (MLVA) was shown to be a promising method for genotyping a number of pathogens 789101112131415. When applicable, MLVA is of great interest, because data produced in different laboratories can be easily exchanged and merged. This is especially relevant in the case of pathogens such as Y. pestis, for which strains cannot be easily exchanged for security reasons. In this context, the availability of standardized and easy to set-up typing tools to facilitate the research efforts on this pathogen is important. The complete genomic sequences of Y. pestis CO92 16, biovar Orientalis, and of strain KIM 17, biovar Medievalis, have been determined, facilitating the identification of tandem repeats and consequently the selection of primers for MLVA 815.

Until now only small series of Y. pestis strains were typed by MLVA 89 and although the method seemed appropriate to genotype reproducibly and accurately, a large scale study seemed necessary. In the present work, a collection of 180 isolates of Y. pestis, from different geographical origins, and including various Y. pestis reference strains were typed using the 25 VNTRs previously described 8.

In the present report, an MLVA typing assay comprising 25 markers has been applied to a collection of Y. pestis isolates of various origins, but with a strong bias towards Orientalis strains from South-East Asia. One hundred and eighty strains or isolates (Table 1) have been genotyped and 61 different MLVA genotypes were identified (see Additional file 1). Clustering analysis and minimum spanning tree analysis suggest relations between the different strains and biovars which are in excellent agreement with current knowledge. In spite of the very limited number of Antiqua and Medievalis strains which could be investigated here, the data obtained suggest the existence of two groups of Antiqua strains. The first group from Russia and Asia represented by genotypes 6, 7 (from Russia) and 8 (KUMA, Yokohama) holds an intermediate position between the Medievalis and the Orientalis group. The second group comprises the African Antiqua strains. IS100 typing distinguishes the KUMA and Yokohama isolates which were typed as A2 and A1b type, respectively 5. They are very similar in DNA microarrays studies 20.

Medievalis and Orientalis strains derive from Antiqua strains by the loss of metabolic functions, respectively the capacity to reduce nitrate, and to metabolize glycerol. Whereas all Orientalis strains investigated so far (and including this report) are derived from a single ancestor carrying a simple deletion in the glpD gene 5, we report here that the Medievalis phenotype can be associated with at least two independent mutation events in the napA gene. This underlines the fact that the initial biochemical tests should be complemented, or replaced, by direct molecular analyses of the glpD and napA genes.

It is tempting to speculate about possible scenarios suggested by Figure 3 and current knowledge. Pestoides strains originate from Central Asia (reviewed in 6), and are proposed to be an outgroup of the Y. pestis group. Their genetic composition is relatively distinct from Y. pestis, in terms of plasmids and chromosome, as assayed by DNA array analysis for instance 20. In Figure 3, the Pestoides strain studied is very distantly related to an hypothetical missing link close to the center of the figure. Hypothetical missing links (open circles) are created by the Minimum Spanning Tree software if they result in a reduction of the total tree length. Many missing links are suggested by the software in this area. They enable the connection of the three branches made of the African Antiqua group, the Medievalis group, and the Orientalis group. The strains closest to this central position are the Antiqua strains from Asia. This and the position of the Pestoides strain suggest that all three biovars may originate from Asia. Indeed, much more strains from these regions will be needed to test this hypothesis. For instance, strain Nicholinsk51 was shown by 5 to share some features of Orientalis strains while being an Antiqua strain from Asia. The authors favored the hypothesis that this strain was a revertant from the Orientalis phenotype. We would rather predict that Nicholinsk51 will be placed by MLVA analysis between the central group of Figure 3 and the Orientalis group.

In total, in our study and that of Klevytska et al. 9, 61 VNTRs were characterized, and still more exist in the Y. pestis genome 15 that can be tested for the selection of an optimal set for MLVA if necessary. Different questions can be addressed by different sets of tandem repeat loci. Global phylogenetic investigations will best be done using loci with a low or moderate mutation rate. Forensics, or local outbreaks investigations, may use loci with a higher mutation rate and particularly simple sequence repeats such as the tetranucleotide described by Adair et al. 21. Mutation rate of a tandem repeat locus and phylogenetic value is very poorly predicted from sequence analysis 1522 so that it has to be experimentally measured by typing collections of isolates, as done here (Table 2 and Additional file 1).

Once set-up, MLVA is a very powerful and reproducible genotyping method and it is hoped that this simple molecular tool will help unravel the molecular phylogeny of Yersinia pestis when being applied to a larger number of isolates. In comparison, MLST analysis 3 proved to be almost non informative within Y. pestis. DNA array analysis 2023 shows a very low resolution with only a few different genotypes identified so far. IS typing by Southern blotting has a very high discriminatory power, but the resulting data is not easily comparable between different laboratories. PCR-IS typing developed by Motin et al. 5 provides exchangeable data but with a much reduced resolution as compared to classical IS typing. MLVA typing may thus turn out to be the method of choice for Y. pestis, once more isolates will have been typed, common genotype databases put together, and reference collections of markers selected. As one step in this direction, the data has been made accessible on our Web site http://bacterial-genotyping.igmors.u-psud.fr. This includes not only the full dataset which can be recovered from Additional file 1, but also the possibility to run queries with new MLVA data. This may be of use for investigators lacking the specialized tools or expertise required to run MLVA clustering analyses. A very satisfying typing resolution (but not a robust phylogenetic analysis) can readily be achieved by PCR amplification of only 7 loci.

FAM did most of the typing work and CP and GV did the error checking analysis. CP performed the biovar assignment by molecular tests and the PCR-IS typing. FR was in charge of the CEB strain collection and prepared the DNA samples. HN provided DNA from a number of additional reference strains. GV initiated and managed the project, and was in charge of the Bionumerics database and clustering analyses. CP, HN, and GV wrote the report. All authors read and approved the final manuscript.