Molecular characterization of endemic salmonella infections ... - Evira

4.1.3 S. Typhimurium DT1 isolates (IV)
The isolates (total n = 255) were obtained from the National Public Health Institute, Helsinki,
Finland (n = 140) and from the Finnish Food Safety Authority Evira (EELA is a part of it), Helsinki,
Finland (n = 115). The isolates were obtained from humans, animals, feed, or the environment
from 1972 to 1999. They were chosen among all available isolates of S. Typhimurium DT1 based
on details of origin (animal species, date or year of isolation, geographical location) in order to get
as representative a material as possible. In practice the isolates had to be widely distributed both
in time and location. The isolates from humans from 1972 to 1999 were classified either as
domestic (a patient had not been abroad during the month preceding the time when the specimen
was taken) or foreign based on the recent travelling history of the patient. However, in judging
whether the origin of an isolate is domestic or foreign in origin, there is always the risk of
misclassification. Most of the isolates of foreign origin were from countries to which tourism from
Finland is common. Travellers might have already had the Salmonella bacteria when leaving home
for the respective destination. The Salmonella isolation might have been carried out after returning
to Finland, and the isolate threfore classified as ‘foreign’ based on the basis of recent foreign
travel. Such a misclassification might be easier to detect as S. Typhimurium DT1 is rare as an
infection source in many countries. The domestic isolates were isolates obtained from outbreaks
(for the period 1972 to 1988). The other isolates were obtained from outbreaks or sporadic cases
(1990 to 1999). In either case, all isolates had been serologically confirmed to be S. Typhimurium,
phage typed as DT1 and stored at -70°C, except for the isolates obtained in the 1980s, which had
been stored on egg agar slopes prior to analysis.
4.2 METHODS
4.2.1 Pulsed-field gel electrophoresis (PFGE) (I-IV)
Preparation of PFGE-samples (I-IV). Chromosomal DNA was prepared in gel blocks as described
previously by Birren and Lai (1993), but with some modifications. Bacteria were harvested from 1.1
ml of an overnight culture. Agarose (1% low melting) was used for the plugs (InCert Agarose, FMC
BioProducts, Rockland, ME, USA). Lysozyme treatment at 37°C for 4 hrs was followed by
proteinase K at 50 °C for 20 hrs. The plugs were stored in 0.5 M EDTA at 4 °C.
XbaI and S1-nuclease digestion for PFGE (I-IV). The agarose plugs were dialysed against 10 mM
Tris, pH 7.4, 1 mM EDTA, 50 mM NaCl before treatment. Restriction enzyme and S1-nuclease
analyses were performed using slices from the same plugs. The slices were digested at 37°C for
16 - 18 hrs, with 20 units of XbaI in the reaction buffer supplied by the manufacturer (New England
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