Evaluation of four molecular typing methodologies as tools for determining taxonomy relations and for identifying species among Yersinia isolates

Abstract

In the last few decades, molecular typing has become an important tool in taxonomic, phylogenetic and identification studies of numerous groups of bacteria, including the yersiniae. In this study, Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), Pulsed-Field Gel Electrophoresis (PFGE), 16S rRNA gene sequencing and Multilocus Sequence Analysis (MLSA) were performed to determine the ability of these techniques to be used in taxonomy and identification of Yersinia strains. A total of 60 Yersinia strains were genotyped by ERIC-PCR and PFGE. Moreover, an in silico analysis was carried out for 16S rRNA gene sequencing and MLSA, using 68 and 49 Yersinia strains, respectively. A phylogenetic tree constructed from the ERIC-PCR, 16S rRNA gene sequencing and MLSA data grouped most of the Yersinia species into distinct species-specific clusters. In the PFGE assay these clusters were not observed. On this basis, ERIC-PCR, 16S rRNA gene sequencing and MLSA seem to be valuable techniques for use in taxonomic and identification studies of the genus Yersinia, whereas PFGE does not. Furthermore, ERIC-PCR has the advantage of being a cheaper, easier and faster assay than 16S rRNA gene sequencing or MLSA, and for these reasons can be considerate an alternative tool in taxonomic studies of yersiniae.

Molecular typing of Yersinia pseudotuberculosis strains isolated from livestock in Brazil

“…Despite the fact that several reports regarding the genotypic diversity of Yersinia species, including Y. pseudotuberculosis strains isolated around the world have been published, the molecular epidemiology of Y. pseudotuberculosis strains isolated from livestock in Brazil remains unclear (Kim et al, 2003;Voskressenskaya et al, 2005;Martins et al, 2007;Souza et al, 2010;Ch'ng et al, 2011;Laukkanen-Ninios et al, 2011).…”

“…A detailed protocol for the MLST procedure, including the primers used for the amplification of the seven housekeeping genes and the annealing temperature is available in the Y. pseudotuberculosis MLST database (http://mlst.ucc.ie/mlst/dbs/Ypseudotuberculosis). All amplifications were performed in a total volume of 50 µL as described by Souza et al (2010). A sample of the complete mix without the DNA was used as a negative control in all runs.…”

“…The ERIC-PCR assay was performed as described by Souza et al (2010) using the primer pair described by Versalovic et al (1991). The ERIC-PCR assay was repeated twice for each strain to verify the reproducibility of the results.…”

“…The genomic DNAs of all strains were prepared in agarose plugs as described by Souza et al (2010). The plugs were digested with 40 U XbaI (Invitrogen) for 18 h. Macrorestriction fragments were resolved by contour-clamped homogeneous electric field electrophoresis in a CHEF DR ® III Pulsed Field Electrophoresis System (Bio-Rad Laboratories, Berkeley, CA, USA) with an electric field of 6 V/cm and an angle of 120°.…”

Facilitated Molecular Typing of Shigella Isolates Using ERIC-PCR

“…The technique requires no specialized equipment or reagents in a laboratory with PCR capabilities. Because the number of organisms that are successfully typed with ERIC-PCR continues to grow to include diarrheogenic 22 and uropathogenic E. coli, 23 Pseudomonas aeruginosa, 24 Aeromonas hydrophila, 25 Vibrio parahemolyticus, 26 Stenotrophomonas, 24 Klebsiella pneumoniae, 27 Yersinia enterocolitica, 28 Bartonella henselea, 29 and Helicobacter pylori, 20 the advantage of adopting this technique at the hospital and regional level also increases. Experience with this simplified technique allows for the preliminary investigation of many important nosocomial or regional outbreaks without the need for a disease-specific approach after primary culture.…”