Biology Reference
In-Depth Information
dNTP (Fermentas), primer (from 0.2 to 1
l
AmpliTaq DNA polymerase (Fermentas), and 4 mM MgCl
2
(Fermentas) were
added to the extracted DNA (from 1 to 5
M, Sigma Genosys), 3 U/50
m
m
l).
A pair of primers, Flic-s/Flic-as (Lim et al.
2003
), was used for the identification
of the
flicC
gene in
S. typhimurium
serotypes. Pathogenic gene fragments were
identified through amplification with the following pairs of primers: ST 139/ ST
141 (
inv
A gene
,
284 bp), 337 L/338 R (
pho
P gene, 299 bp), SpvC for/SpvC rev
(
spv
C gene, 699 bp), and SpvR 1/SpvR 2 (
spv
R gene, 891 bp) (Rhan et al.
1992
;
Way et al.
1993
; Abouzeed et al.
2000
; Haneda et al.
2001
). Pulsed-field gel
electrophoresis (100 V, 498 mA) was performed in a 2% agarose gel (Eppendorf)
for 2 h.
m
25.3 Results
The values of the psycrophilic aerobic count in the 21 samples of ice fish ranged
between 2.62 log CFU/g and 6.47 log CFU/g (mean value: 4.75 log CFU/g), while
values in the relative defreezing waters fluctuated between 3.87 log CFU/ml and 7
log CFU/ml (mean value: 5.14 log CFU/ml). The mesophilic aerobic count in the
fresh fish varied between 2.91 log CFU/g and 8.61 log CFU/g, with a mean value of
5.86 log CFU/g (Fig.
25.1
). This last result depended on the geographic origin: 5.49
log CFU/g (Adriatic Sea), 6.12 log CFU/g (Tyrrhenian Sea), and 4.97 log CFU/g
(Ligurian Sea). The MPN method highlighted
E. coli
contamination in 23.8% of the
samples of ice fish, 14.3% of defreezing waters, and 7.0% of fresh fish. Two
samples (PG 6 and PG 16) were negative using the MPN method, but
E. coli
was
identified from selective media used in the isolation of
S. enterica
and
V. parahaemolyticus
. No samples were positive for
V. parahaemolyticus
and
L. monocytogenes.
L. grayi
was isolated from 8% of the samples,
L. ivanovii
from 3.5%, and both
L. innocua
and
L. welshimeri
from 2.3% of the samples.
S
.
napoli
, sensitive to all
tested antibiotics, and
S
.
typhimurium
, resistant to streptomycin, ampicillin, chlor-
amphenicol, and sulfonamide, were isolated, respectively, from two samples of ice
fish (PG 15 and PG 21). For the first serotype, only genes
invA
and
phoP
were
identified, while
S
.
typhimurium
possessed the
fliC
gene (Fig.
25.2
), which
5.14 log CFU/ml
5.86 log CFU/g
Fig. 25.1 Mean value of the
mesophilic and psycrophilic
aerobic counts of fresh fish,
ice fish, and defreezing water
4.75 log CFU/g
Fresh fish
Ice fish
De-freezing water