Genotyping of Yersinia strains by high-resolution melting analysis

Author(s)

Roberto Antonio de Souza

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2013-05-23

Examining board members

Juliana Pfrimer Falcão; Alzira Maria Paiva de Almeida; Marcelo Brocchi; Ana Lucia da Costa Darini; Rodrigo Tavanelli Hernandes

• Advisor: Juliana Pfrimer Falcão

Abstract

The genus Yersinia belongs to the family Enterobacteriaceae and comprises 17 species. Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are well recognized human and animal pathogens. Y. pestis causes plague. Y. pseudotuberculosis and Y. enterocolitica are, usually, causative agents of food-waterborne gastroenteritis. The other 14 Yersinia species are considered to be non-pathogenic, with the exception of Y. ruckeri serogroup O:1 which causes infections in fishes. In the last few decades, molecular typing has become an important tool in phylogenetic studies of several microorganisms and the development of fast and inexpensive typing systems can facilitate epidemiological studies of bacterial infections. The present study aimed to develop a method of Yersinia spp. genotyping based on high-resolution melting analysis (HRMA) in order to differentiate the single-nucleotide polymorphisms (SNPs) present in the 16S rRNA, glnA, gyrB, hsp60 and recA sequences and apply it in the typing of 40 Y. pseudotuberculosis strains and 50 Y. enterocolitica strains, as well as, to separate by HRMA the Y. pseudotuberculosis and Y. enterocolitica species. The SNPs were determined in the sequences of the aforementioned loci using a set of 119 Yersinia strains deposited in the GenBank/EMBL/DDBJ database. It were found in the gene sequences analyzed of Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii and Y. ruckeri 10, 10, 9, 6, 4, 1 and 1 SNPs, respectively. No SNPs was found in the analyzed sequences of Y. pestis and a large number of SNPs were found in the analyzed sequences of Y. frederiksenii, Y. kristensenii and Y. massiliensis what prevented their genotyping by HRMA. The remaining Yersinia species were not analyzed. It was designed primer pairs to flank the SNPs found in each Yersinia species tested. Using a specie-specific set of primers, the genetic diversity of each Yersinia species used was determined by HRMA and the phylogenetic analysis was based on the concatenated sequence composed by the nucleotides identified in each fragment analyzed. Clustering was performed with the software package BioNumerics using UPGMA method and 1,000 bootstrap replicates. The phylogenetic tree constructed for Y. pseudotuberculosis grouped the strains into bio-serogroups specific clusters. The strains of 1/O:1 bio-serogroup were grouped into one cluster and the strains of 2/O:3 bio-serogroup into iv other cluster. The phylogenetic tree constructed for Y. enterocolitica grouped the strains in three clusters. The highly pathogenic strains, of biotype 1B, were grouped into one cluster, the moderate pathogenic strains, of biotypes 2, 3, 4 and 5, were grouped into a second cluster and, the non-pathogenic strains, of biotype 1A, were grouped into a third cluster. The clusterization of Y. pseudotuberculosis and Y. enterocolitica were consistent with the pathogenic profile characteristic of these two Yersinia species. No significant epidemiological correlation was found in the grouping of Y. bercovieri, Y. rohdei, Y. intermedia Y. mollaretii and Y. ruckeri according to HRMA results. Moreover, the HRMA-based method develop here was able to separate the Y. pseudotuberculosis and Y. enterocolitica species. The HRMA assay developed in this study can be used as an alternative for the genotyping and the differentiation of Y. pseudotuberculosis and Y. enterocolitica. This method can also complement sequence-based methods and facilitate epidemiological studies of these two Yersinia species. (AU)

• FAPESP's process: 09/08455-1 - Genotyping of Yersinia ssp. strains by high-resolution melting analysis
• Grantee: Roberto Antonio de Souza
• Support Opportunities: Scholarships in Brazil - Doctorate