Genetic Diversity of Food-Isolated Salmonella Strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR)

Abstract

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

ERIC-PCR genotyping of field isolates ofSalmonella entericasubsp.entericaserovar Gallinarum biovars Gallinarum and Pullorum

“…ERIC-PCR has been shown as a powerful tool for demonstrating variability among isolates (Sachdeva & Virdi, 2004;Fendri et al, 2013). It has been suggested that ERIC sequences would in all likelihood have some functional role in the bacterial cell (Asinmov et al, 2005;De Gregorio et al, 2005) although this has not yet been demonstrated.…”

“…Only DNA samples which showed quality parameters of approximately two were used in this study. Fendri et al (2013) with the modifications described below. Each PCR reaction was made up of 1x DreamTaq TM Green Buffer with MgCl 2 (Thermo Fisher Scientific Inc., Asheville, NC, USA -EP0711), 1.0 pmol of primers ERIC1R (5′-ATG TAA GCT CCT GGG GAT TCA C-3′) and ERIC2 (5′-AAG TAA GTG ACT GGG GTG AGC G-3′) (Sigma-Aldrich, Saint Louis, MO, USA), 5 mM of MgCl 2 (Invitrogen Corp., Carlsbad, CA, USA), 240 µM of each deoxynucleotide triphosphate (dNTP) (Invitrogen Corp.), 5 µl of DNA template (approximately 60 ng), 2 U of Taq DNA polymerase (Thermo Fisher Scientific Inc.) and ultrapure water (Sigma-Aldrich) to a final volume of 25 µl.…”

“…The Enterobacterial Repetitive Intergenic Consensus (ERIC) technique has been used in order to identify genetic diversity in enterobacteria (Versalovic et al, 1991;Sachdeva & Virdi, 2004;Yu et al, 2011;Fendri et al, 2013;Dorneles et al, 2014). This method has allowed separation of clonal strains by the detection of specific chromosomal segments (ERIC sequences) suggesting this could be applied in epidemiological investigations (Dorneles et al, 2014).…”

“…However, PFGE is both time consuming and requires staff skilled in the method to be reproducible. The ERIC method, generally considered to be both reliable and sensitive, is a much simpler procedure than PFGE (Kosek et al, 2012;Fendri et al, 2013;Dorneles et al, 2014).…”

Sequence-Level and Dual-Phase Identification of Salmonella Flagellum Antigens by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

“…Recently, multilocus enzyme electrophoresis and multilocus sequence typing have been explored to link serotypes with housekeeping gene patterns (18)(19)(20), but neither method agreed well with results obtained by the traditional serotyping method. The bacterial identification method using whole-genome restriction patterns, pulsed-field gel electrophoresis, has been applied to some common serovars of Salmonella but could not achieve the quality of serotyping data because of variable patterns, even among strains with the same serovars (21). Lastly, MS techniques, especially matrix-assisted laser desorption ionization-time of flight MS, have been used to type Salmonella in recent years because of their speed and ease of use (22,23).…”