Pulsed-Field Gel Electrophoresis, Conventional, and Molecular Serotyping ofListeria monocytogenesfrom Food Proficiency Testing Trials Toward an Harmonization of Subtyping at European Level

Abstract

The European Union Reference Laboratory for Listeria monocytogenes (EURL for L. monocytogenes) coordinates a European network of 29 National Reference Laboratories (NRLs). Depending on a national decision, NRLs undertake food, environmental, and veterinary L. monocytogenes strain surveillance in their respective countries. In the framework of the PulseNet Europe network, two pulsed-field gel electrophoresis (PFGE) subtyping proficiency testing (PT) trials were carried out in 2003 and 2006. The obtained data showed that PFGE profiles can be compared and exchanged between laboratories. However, no further PT trial had been performed since 2006. In this context, two PT trials were organized by the EURL to evaluate the ability of NRLs to perform conventional serotyping, molecular serotyping and PFGE subtyping. Eleven well-characterized isolates of L. monocytogenes were used: six and nine isolates were tested in 2009 and 2010, respectively. Three isolates were repeated between the two studies. In the 2010 panel, a strain was tested in duplicate, and two strains were related to the same epidemiological group. The strains were analyzed blind in different laboratories (17 in 2009 and 25 in 2010) using (1) their own in-house method for serotyping methods and (2) standardized protocols based on the PulseNet protocol for PFGE. For conventional serotyping, 86.0% in 2009 and 91.0% in 2010 of the serotypes obtained were in agreement with the EURL data. For molecular serotyping, 93.5% of the results in 2009 and 95.2% in 2010 matched the EURL data. For PFGE, 68.9% in 2009 and 81.7% of the combined AscI/ApaI profiles were indistinguishable from the EURL reference profiles. The variations observed could be attributed to slight standardization defaults or, in a few cases, to a failure in DNA extraction. These PT trials provided a valuable opportunity to improve the subtyping ability of NRLs and facilitate exchanges of subtyping data in the future.

Characterization of epidemic clones of Listeria monocytogenes serotype 4b isolated from humans and meat products in Brazil

“…Cluster analysis and dendrogram construction were performed using BioNumerics software version 4.0 (Applied Maths, Belgium), using the UPGMA method and the Dice coefficient with a 1.5% tolerance index. A minimum of 97% similarity was established to define a particular clone profile, as proposed by Félix et al [21]. The Simpson diversity index was used to evaluate the discriminatory ability of PFGE [22].…”

Scientific Opinion on the evaluation of molecular typing methods for major food-borne microbiological hazards and their use for attribution modelling, outbreak investigation and scanning surveillance: Part 2 (surveillance and data management activities)

“…Joint EQA studies on molecular typing characterization of isolates by PFGE have been recently organized by the EU-RLs for L. monocytogenes, Salmonella and E. coli including VTEC, together with the corresponding laboratory appointed by the ECDC to coordinate the public health laboratory network (ECDC, 2013a(ECDC, , 2014a(ECDC, , 2014bEU Reference Laboratory for E. coli, 2013;Felix et al, 2012;Felix et al, 2013). EQA studies were carried out using the reference protocol and evaluation criteria in use in the PulseNet International and PulseNet Europe networks (PulseNet International, online-a, online-b, online-c).…”

Pulsed-Field Gel Electrophoresis Proficiency Testing Trials: Toward European Harmonization of the Typing of Food and Clinical Strains ofListeria monocytogenes

“…Three strains-5, 6, and 7-were selected, since most of the failures occurred for these strains during the 2009 PT trial (Félix et al, 2012b). Strain 10 was tested in duplicate (not shown in Table 1 or Fig.…”

“…Between 2007 and 2012, the EURL regularly organized typing training sessions, annual workshops, and Proficiency Testing trials (PT trials) on serotyping and pulsed-field gel electrophoresis (PFGE) typing. In particular, the EURL encouraged NRLs to use a harmonized method for PFGE (Félix et al, 2012b) and a Standard Operating Procedure (SOP) (Félix et al, 2012a) for the analysis of gels and interpretation of the PFGE profiles obtained so that NRLs can compare them and exchanges can be made between NRLs and the EURL.…”

“…For the NRLs, the EURL organized two PT trials in 2009 and 2010 (Félix et al, 2012b). However, as these PT trials were focused on the PFGE protocol, the NRLs did not yet have the opportunity to be evaluated on their ability to interpret the PFGE profiles, according to the SOP (Félix et al, 2012a).…”