2.2. Listeria species isolation
The ISO 11290-1 method for enrichment of Listeria was used for
samples taken in this study, and all Listeria species detected were
included in the subsequent analysis (ISO, 2002). Brilliance Listeria
chromogenic agar (Oxoid, UK), RAPID’L.mono agar (Bio-Rad, UK),
and Oxford Listeria selective agar (Oxoid, UK) were used to plate
enriched samples, and presumptive Listeria isolates were
confirmed using the Microbact Listeria identification system
(Oxoid, UK). Four Listeria species were tested from every positive
sample, to account for (possible) strain diversity in individual
samples. All samples were processed in the laboratory on the same
day they were sampled.
2.3. PFGE sub-typing of Listeria isolates
Sub-typing of Listeria isolates was performed using pulsed-field
gel electrophoresis (PFGE) following the standard PulseNet protocol
(PulseNet USA, 2009), with the following modifications: sodium
dodecyl sulfate (SDS) was not included in the 1% SeaKem Gold plug
agarose; electrophoresis was performed on a CHEF Mapper for 21 h.
The restriction enzyme AscI was used to generate the PFGE profiles.
PFGE profiles were analyzed using BioNumerics V5.1 (Applied
Maths, Belgium), using the Dice coefficient and UPGMA analysis,
with ‘Optimization’ and ‘Tolerance’ settings of 1%. For the purposes
of this study, a ‘strain’ was definied as having a unique PFGE
fingerprint, or sub-type.