In this study a draft genome sequence of Y. enterocolitica
4/O3 type strain Y11 was used as a template for the search for VNTRs. Twelve VNTR marker loci were initially selected for genotyping. However, only six loci met the criteria for use for MLVA. It is noteworthy that the same number of marker loci allowed outbreak and sporadic Escherichia coli
O157:H7 isolates to be distinguished (23
). This low number of loci is usually sufficient for outbreak investigations and strain clustering (29
), while an analysis based on multiple loci is recommended for phylogeny studies (18
Any method able to estimate the PCR product size or to distinguish the allele length with a sufficient resolution with respect to the repeat unit size can be applied for MLVA. The currently used techniques are agarose gel electrophoresis, DGE, and DNA sequencing (13
). Although the first method is cheap and easy to perform, it was found to be unreliable. The automated sequencing technology is highly reproducible and effective, but it requires expensive equipment and reagents. For this reason, DGE (13
), which has a high resolution, which is reproducible, and for which the cost of reagents is low, was used. However, our MLVA can also be used in laboratories equipped with an automated DNA sequencer.
Although DGE is considered a method that allows the determination of DNA length with the accuracy of a single nucleotide, a high degree of reliability may be achieved only when the concentrations of acrylamide and bisacrylamide as well as the migration distance are optimized (26
). In this study, the use of a gel concentration of 8% and a migration distance of 40 to 50 cm was proposed for the optimal separation of all MLVA marker amplicons in a single gel. The only disadvantage of applying uniform electrophoresis conditions that was observed was a minor inaccuracy of the DNA size compared to the amplicon size determined by DNA sequencing. Interestingly, repeated DGE analyses showed that this inaccuracy was reproducible. Lista and colleagues (19
) observed the same phenomenon using an automated DNA sequencer for the separation of VNTR locus amplicons for B. anthracis
MLVA genotyping. Those authors recommended the use of a tandem repeat copy number or a size calculated from DNA sequencing for cluster analysis and genotype determination. Therefore, in order to support conformity with the results obtained in laboratories equipped with DNA sequencers, the VNTR marker size estimated by DGE was converted to the size determined by sequencing (Table ).
Still, the majority of various genotyping techniques applied to Y. enterocolitica
require the use of reference strains to ascertain the consistency of interlaboratory results. In contrast, MLVA is determinative and no reference strain is needed for calibration. A local reference strain from which amplicons have been sequenced is sufficient for gel-to-gel comparison. Since the results of MLVA are integers, the genotypes determined in different laboratories may be easily compared and joint databases can be produced, as shown in various studies (18
). MLVA demonstrated a high discriminatory power not only for Y. enterocolitica
bioserogroup 4/O3 strains of worldwide origin but also for those strains isolated within a country or even within a hospital. In this study, 20 MLVA genotypes were distinguished in 21 nonepidemiologically linked strains of Y. enterocolitica
4/O3 from Poland. Buchrieser and colleagues, who tested 17 bioserogroup 4/O3 isolates from Austria, distinguished nine genotypes when the PFGE results for NotI and XbaI were combined (8
). Among 17 of the 4/O3 strains from England and Wales, fluorescent amplified fragment length polymorphism analysis distinguished 13 genotypes (10
). The best resolution for bioserogroup 4/O3 reported so far was obtained by PFGE with three endonucleases. This method distinguished 30 genotypes among 128 strains isolated worldwide (11
). Overall, the data showed that on a countrywide scale, MLVA would offer a higher discriminatory power for Y. enterocolitica
4/O3 than the methods mentioned above. On the other hand, Noller and colleagues (24
) concluded that the high resolution of MLVA may be in part a result of a genetic noise produced by the VNTR loci which are hypervariable. Three of the seven VNTR loci used by those authors for discrimination of sporadic and epidemic E. coli
O157:H7 isolates mutated after multiple serial passages under laboratory conditions. One of them, TR2, indicated hypermutability. Similar phenomena were observed in our study. Although the seven VNTR loci selected at the beginning were stable during multiple serial passages in the laboratory, a comparison of the MLVA genotypes of epidemiologically linked isolates showed that they exhibited a single variation in the V2a locus and hypermutability of the V10 locus. Therefore, the latter locus was excluded from the MLVA scheme that was developed, while the six remaining loci were finally considered reliable because of their genotype stability, which is required for routine manipulations.
Although this study was conducted with a limited number of epidemiologically linked isolates, it is noteworthy that our results support the generalizations made by Noller and colleagues (24
) about the interpretation of MLVA data during outbreak investigations. Briefly, among a group of epidemiologically linked strains, isolates indistinguishable by MLVA originated from the same source, while isolates indicating diversity in a single or a double tandem repeat in a single VNTR locus came from a point source. If an isolate differs at two VNTR loci, a degree of variation in these loci needs to be considered. Taken together, the data presented in our study and those previously reported by Noller and colleagues (24
) demonstrate that each MLVA scheme addressed for epidemiological investigations must be tested with a group of epidemiologically linked strains.
It is noteworthy that the VNTR markers designed for Y. enterocolitica subsp. palearctica O3 were also present in strains of other pathogenic serogroups, i.e., O5,27 and O9. Despite the low number of strains tested, a high degree of diversity of genotypes was observed, as each strain tested had its own genotype. In addition, two VNTR markers, V5 and V7, were found to be present in the American Y. enterocolitica subsp. enterocolitica serogroup O8 strains (data not shown), indicating their potential usefulness for the genotyping of isolates belonging to this highly pathogenic group.
The use of different genotyping techniques, including the method considered to be the “gold standard” for the subtyping of Y. enterocolitica
O3 (PFGE), showed that isolates originating from an outbreak or from a given geographical area were often so homogeneous that they could not be differentiated (11
). Considering the wide spectrum of loci analyzed by a variety of genotyping methods, strains of Y. enterocolitica
O3 were supposed to be highly clonal (1
). Correspondingly, the relatively high number of MLVA genotypes observed in this study could argue for support of the assumption that the Y. enterocolitica
O3 VNTR loci evolved at a faster speed than that which occurs on the evolutionary scale (29
) and therefore does not argue against the clonal nature of Y. enterocolitica
O3. As a result of the high-speed evolution of VNTR loci, a convergence of single VNTR markers may appear even in strains of distant geographic origins. To reduce the influence of such convergence, more than one VNTR marker must be used for genotyping (18
Due to its high discriminatory power, MLVA appears to be a helpful tool for distinguishing Y. enterocolitica subsp. palearctica isolates which are difficult to differentiate by PFGE but which are suspected of being epidemiologically unrelated. In addition, as was shown for the blood transfusion-related strains used in our study, the MLVA genotype may serve as a fingerprint for epidemiological investigations.
In summary, the MLVA genotyping tool developed in this study was capable of distinguishing 45 genotypes among 62 isolates of Y. enterocolitica 4/O3 of worldwide origin tested. Our results showed that the clonal structure of Y. enterocolitica O3 was highly diverse in the VNTR loci. The MLVA genotyping technique described in the present study appears to be a promising tool for epidemiological investigations of Y. enterocolitica O3 outbreaks.