Multiple-Locus Variable-Number Tandem-Repeat Analysis of Pathogenic Yersinia enterocolitica in China

The predominant bioserotypes of pathogenic Yersinia enterocolitica in China are 2/O: 9 and 3/O: 3; no pathogenic O: 8 strains have been found to date. Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) based on seven loci was able to distinguish 104 genotypes among 218 pathogenic Y. enterocolitica isolates in China and from abroad, showing a high resolution. The major pathogenic serogroups in China, O: 3 and O: 9, were divided into two clusters based on MLVA genotyping. The different distribution of Y. enterocolitica MLVA genotypes maybe due to the recent dissemination of specific clones of 2/O: 9 and 3/O: 3 strains in China. MLVA was a helpful tool for bacterial pathogen surveillance and investigation of pathogenic Y. enterocolitica outbreaks.


Introduction
Yersinia enterocolitica is a gram-negative entero-pathogen causing gastroenteritis, especially in children [1]. Human clinical infections with Y. enterocolitica occur after ingestion of contaminated food, water, or by direct inoculation through blood transfusion. Infection with Y. enterocolitica causes several clinical manifestations e.g. diarrhea, enteritis, enterocolitis, mesenteric lymphadenitis and some complications and sequelae, reactive arthritis, and erythema nodosum [2]. Lots of animal reservoirs are found for Y. enterocolitica, including swine, monkeys, dogs, rabbits, deer, and birds; swine is the predominant carrier [3]. Some research has shown high carrier rates of Y. enterocolitica in the swine oral cavity [4].
The distribution of pathogenic Y. enterocolitica is diverse in China and all strains belong to serotypes O: 3 and O: 9. These two serotypes can be isolated from livestock and poultry where swine are the primary reservoir [7]. The biotype distribution of Y. enterocolitica is different worldwide and also in the provinces of China. The predominant bioserotype in the world is 4/O: 3, especially in most European countries and Japan; while in America, bioserotype 1B/O: 8 strains once predominated, then substituted by serotype O: 3 [7]. The bioserotype distribution of Y. enterocolitica in China is different from other countries, predominantly 2/O: 9 and 3/O: 3, fewer 4/O: 3, and no pathogenic 1B/ O: 8 strains [8].

Simpson's index of diversity
The Simpson's index of diversity was calculated using BioNumerics software [23]. For the total strains, V5 exhibited the highest discriminatory power (DI = 90.43%), resolving 18 different alleles; the least variation was observed for locus V4. However, for the strains isolated from Ningxia province, V10 exhibited the highest discriminatory power (DI = 84.44%); while the most amount of alleles (14) was located in V5. The least variation also was located in the V4 locus. All of the VNTR loci showed high discriminatory power except for V4 (Table 1).

MLVA analysis of all strains
218 pathogenic Y. enterocolitica were divided into 104 MLVA genotypes (Fig. 1). We found strains that were isolated from the same province and same year where different reservoirs were classified to one MLVA genotype; this suggests an epidemiological link (especially for the Ningxia strains from 1997 to 1999). There was a correlation between the MLVA genotypes and serotypes. Both O: 3 and O: 9 serotype strains (except for a few) were separated into two MLVA genotyping clusters. O: 9 strains were primarily found in Ningxia, Jilin, and Henan provinces; while, the O: 3 strains were diversely distributed in different provinces in China. Four of the five strains isolated from Japan were classified to O: 3 serotype cluster according to their MLVA genotypes. This was in accordance with the serotype characteristic in China.
The strains isolated from a patient in Jiangsu Province in 1994 had the same MLVA genotype as the strains isolated from dogs in 2004 where we previously showed this linkage [24]. The patient's strain had the same PFGE profile (K6GN11C30021 PFGE-NotI profile) as the strains isolated from dogs in 2004 and 2007; however, here we showed that the isolates from patient and dogs in 2004 had the same MLVA genotype, while the strains isolated from dogs in 2007 displayed the different MLVA genotype. Therefore, MLVA may have a higher discriminatory power than PFGE. We also compared the MLVA and PFGE results of the seven bioserotype 3/O: 3 isolates from Henan province in 2006 and showed four MLVA genotypes and two PFGE NotI profiles (Fig. 2). The asterisks marked in figure 1 were analyzed strains in figure 2.

MLVA analysis of the Ningxia strains
Among the total strains, 148 pathogenic Y. enterocolitica were isolated from Ningxia Province during 1997 to 1999. MLVA was able to distinguish 51 genotypes among the 148 pathogenic Y. enterocolitica (Fig. 3). All of the strains were bioserotype 2/O: 9 (except four bioserotype 3/O: 3). MLVA genotyping was able to identify strains isolated from the same source, especially for swine (SA98-1286 to SA-286-2) and mouse (IE1763F to IE1872F). Different MLVA genotypes were observed for strains isolated from the same source during different years, which shows the high discriminatory power of MLVA. We constructed a minimum spanning tree (MST) using MLVA data (Fig. 4). Most bioserotype 2/O: 9 strains were clustered in a large MLVA genotyping group (pink area) where only a few 3/O: 3 (BJ and BN) were distant from the center. Two cluster groups were observed among the strains, one was AB and AI (red area) for the strains isolated from mouse in 1998; another was AZ, AU and A2 (blue area) for the strains isolated from swine during 1997 to 1998.

MLVA analysis based on six VNTR loci
In Europe, MLVA with six VNTRs markers was used to genotype Y. enterocolitica ssp. palearctica [15,16]. This assay distinguished outbreak-related and sporadic isolates. To compare the genetic diversity of Y. enterocolitica in China and Europe, we analyzed the data with six VNTR loci. 90 MLVA6 genotypes were distinguished among 218 pathogenic Y. enterocolitica. O: 3 and O: 9 serotype strains were also separated into two MLVA6 genotyping clusters. O: 9 strains were mainly found in Ningxia, Jilin, and Henan provinces; while, the O: 3 strains displayed genotype heterogeneity. However, the isolates from the same province were clustered to same MLVA6 genotype for all the strains, which showed the relatedness with geographic distribution, especially for Ningxia province. The cluster results based on six VNTR loci were almost identical to MLVA genotype distribution applied seven VNTR loci for pathogenic Y. enterocolitica in China.  MLVA analysis of highly pathogenic and nonpathogenic Y. enterocolitica

Analysis of sequences flanking tandem repeats
We performed the same analysis using bioserotype 1B/O: 8 strains from abroad and 19 nonpathogenic strains isolated from China. The data showed only V2A, V4 and V5 loci amplified the differential straps for these strains; whereas V6, V7, V9 and V10 loci showed no results.

Discussion
In Gierczynski et al. research [15], MLVA based on six loci was able to distinguish 76 genotypes among 91 Y. enterocolitica isolated from worldwide origins and 41 genotypes among 51 nonepidemiologically linked bioserotype 4/O: 3 isolates. Only a slight correlation comparing the MLVA genotypes with geographic distribution of the isolates and a minor correlation comparing the MLVA genotypes with serogroups were observed in their study. Our research was based on continuous surveillance of Y. enterocolitica in China over a long period where we considered the similarity of the isolates from the same province. We included the V10 locus in our analysis and distinguished 104 genotypes among 213 pathogenic Y. enterocolitica isolated from China and five isolates from Japan. Most of the isolates were bioserotype 2/O: 9 and 3/ O: 3 where the O: 3 and O: 9 serogroups were separated using MLVA genotyping.
This was an important difference between MLVA-genotypes distribution of Y. enterocolitica subsp. palearctica in China and Europe. The majority of strains tested in Europe belonged to unique genotype, few strains with no known epidemiological link belonged to the same MLVA genotype, such strains were isolated within the same country (Hungary, Poland, and Germany) in the reference ''(15)''; while, our results indicated that the isolates from the same province of China were clustered to the same MLVA6 genotype, which showed the relatedness with geographic distribution. There was also correlation between the MLVA6 genotypes and serogroups in our research. The limited genetic diversity of MLVA results possibly reflected the recent dissemination (in evolutionary scale) of specific clones of Y. enterocolitica 2/O: 9 and 3/O: 3 strains in China.
We also performed the MLVA analysis for the highly pathogenic strains from abroad and 19 nonpathogenic Y. enterocolitica isolated in China where only V2A, V4, and V5 loci showed differential results. Gulati et al. [25]  The distribution, characters, and functions of seven VNTR loci used in our study were not described in the reference ''(15)'', therefore we analyzed the seven VNTR loci using Blastn and Blastp searches in the NCBI data base. The results show V2A is a sensory histidine kinase; V4 is a putative glycerate kinase; V5 is a putative hydrogenase isoenzymes formation protein; V6 is a hypothetical protein; V7 is a putative GTP-binding protein; V9 is a transcriptional regulator; and V10 is a hypothetical protein.
These loci were generally located in the open reading frame of structural genes. V2A, V4 and V5 loci shared homology with the highly pathogenic bioserotype 1B/O: 8 strain 8081 through blast results; and may explain the reason for the amplifications. However, V6, V7, V9 and V10 loci only exist in weakly pathogenic Y. enterocolitica strains, as confirmed herein.

Bacterial strains
A total of 218 pathogenic Y. enterocolitica strains were used where the number of isolates from patients was 11 and all the isolates were from sporadic yersiniosis cases. 52 strains were bioserotype 3/O: 3; two were 4/O: 3, one was 3/O: 5,27, 157 were 2/O: 9, and six were 3/O: 9. During the 1997 to 1998, we performed a large scale of surveillance for animal reservoirs of Y. enterocolitica in Ningxia province, and isolated lots of pathogenic Y. enterocolitica bioserotype 2/O: 9 strains from mouse and swine. 213 strains were isolated from China and five strains were provided by Dr. H. Fukushima. All of the strains were PCR tested to detect virulence genes ( Table 2).    We selected three highly pathogenic foreign strains and compared them to 19 non-pathogenic strains isolated in China (Table 3).

DNA extraction
The bacteria were inoculated on BHI agar; incubated for 24 hours at 25uC; and the genome DNA of the bacteria were extracted using a DNeasy tissue kit (Qingen, Germany) using the manufacturer's instructions.

PCR analysis and sequencing
Using the primers from the reference method [15], VNTR loci were amplified using the primers at a concentration of 0.2 uM with Taq DNA polymerase (TaKaRa, Japan) in the reaction buffer supplied by the manufacturer in a 20-ul reaction volume. PCR amplifications were performed: initial denaturation at 94uC for 10 min; followed by 35 cycles: denaturation at 94uC for 30 s, annealing at 58uC for 30 s, and extension at 72uC for 30 s; and a final extension at 72uC for 3 min. All of the PCR products were sequenced (TaKaRa); and the fragments of each sequence were merged and aligned using DNAstar software; then we computed the numbers of tandem repeats for each strain.

Cluster analysis
Clustering of the MLVA types was performed using BioNumerics software (ver. 5.10) with categorical coefficient of similarity and UPGMA (the un-weighted pair group method using the arithmetic mean) method to estimate genetic differences. For the higher proportion (67.89%) of the isolated strains found in Ningxia province from 1997 to 1999, we tested induplicate. Then, we analyzed the sequences before or after the tandem repeats for each strain, intended. Note: The bracket next to strain numbers are the strains that have lost the plasmid; *strains from Japan; ST, serotype; and BT, biotype; ail, attachment invasion locus of Y. enterocolitica; ystA, the production of heat-stable enterotoxin in Y. enterocolitica; ystB, encode an enterotoxin present mainly in biotype 1A strains; yadA, the plasmid-borne virulence gene involved in autoagglutination, serum resistance, and adhesion; virF, encode transcriptional activators of the yop regulon in pYV plasmid; rfbc: located in the rfb gene cluster, responsible for the biosynthesis of the O side chain of Y. enterocolitica O: 3 strain. doi:10.1371/journal.pone.0037309.t002