Resumo

Introduction: Yersinia pseudotuberculosis is one of the major pathogenic species of the genus Yersinia. In this work, we aimed to compare Multilocus Sequence Typing (MLST), Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) and Pulsed-Field Gel Electrophoresis (PFGE) techniques for subtyping Y. pseudotuberculosis strains and to assess their discriminatory power. Materials and methods: A total of 40 Y. pseudotuberculosis strains isolated from animals were studied. For MLST, seven housekeeping genes (adk, argA, aroA, glnA, thrA, tmk and trpE) were amplified and sequenced. The consensus sequences were obtained using the ChromasPro 1.41 software package for later analysis online in the Y. pseudotuberculosis MLST database (http://mlst.ucc.ie/mlst/dbs/Ypseudotuberculosis). The combination of seven alleles, one for each target gene, provided the sequence type (ST). For ERIC-PCR, the amplifications were performed with 100ng of DNA template, each dNTP at 0.4mM, 4mM MgCl₂, 1U Klen Taq DNA polymerase, 1X PCR buffer and 20pmol of each universal ERIC-primers. Only bands between 100 and 5000 bp were analyzed. For PFGE, the plugs were digested with 40U of XbaI overnight. Macrorestriction fragments were resolved in a CHEF-DR III apparatus with an electric field of 6V/cm and angle of 120°. The pulse times were increased from 1 to 10s over 29h. Only bands between 48.5 and 194 Kb were analyzed. The construction of the dendrograms of genetic similarity of ERIC-PCR and PFGE was performed by the software package BioNumerics 5.1 using the UPGMA algorithm. The discrimination index of MLST, ERIC-PCR and PFGE was assessed by Simpson’s diversity index. Results: By MLST, all the three Y. pseudotuberculosis strains of bio-serogroup 1/O:1 were classified as ST 42 and all the 37 strains of bio-serogroup 2/O:3 were classified as ST 19. ERIC-PCR and PFGE grouped the Y. pseudotuberculosis strains into two main groups, one composed by strains of bio-serogroup 1/O:1 and the other by strains of bio-serogroup 2/O:3. The DI was 0.959, 0.142 and 0.842 for ERIC-PCR, MLST and PFGE, respectively. Conclusion: The Y. pseudotuberculosis strains studied showed to be genetically very similar. All three techniques are suitable to be applied for Y. pseudotuberculosis subtyping studies and were capable to differ Y. pseudotuberculosis strains into virulence-associated bio-serogoups.