Biology Reference
In-Depth Information
230 bp. Positive enrichments from the PCR screening were submitted to manual
IMS performed using Dynabeads anti-
E. coli
O157, O26, O103, O111, and O145
(DY 71004; Dynal, Oslo, Norway) as per the manufacturer's instructions. After
the IMS, a 50-
l suspension was streaked onto the following media: CT-SMAC
(cefixime tellurite sorbitol MacConkey agar) for the detection of the O157
serotype, CT-RMAC (cefixime tellurite rhamnose MacConkey agar) for the
O26 serotype, and EHLY agar for the O103, O111, and O145 serotypes, and
incubated at 37
C for 24 h. Colonies with typical characteristics were subjected
to phenotypic identification using the API 20 E (20100; bioM´rieux) system.
Furthermore, the cultures of the O157 strain were tested using an
E. coli
O157
Latex test kit (DR 620; Oxoid).
m
27.2.3 Molecular Characterization of the Isolates
Fifty-three strains from the different matrices were subjected to multiplex PCR for
the detection of
stx
1
,
stx
2
, and
eae
(Paton and Paton
1998
; EFSA Report
2010
) using
the primers
stx
1
F(5
0
-ATAAATCGCCATTCGTTGACTAC-3
0
),
stx
1
R(5
0
-AGAA-
CGCCCACTGAGATCATC-3
0
),
stx
2
F(5
0
-GGCACTGTCTGAAACTGCTCC-3
0
),
stx
2
R(5
0
-CGCCAGTTATCTGACATTCTG-3
0
),
eae
AF (5
0
-GACCCGGCACAA-
GCATAAGC-3
0
), and
eae
AR (5
0
-CACCTGCAGCAACAAGAGG-3
0
), which
resulted in the amplification of 180-, 285-, and 384-bp products, respectively.
27.3 Results
According to the preliminary screening test, the overall prevalence of VTEC was
11.1% (14% in sheep and 7.8% in lambs). In particular, 18.9% of the skin, 14.7% of
the carcass, and 10.5% of the mucosa samples were positive (Table
27.1
).
The strains subjected to molecular characterization were (1) CT-SMAC, n.24,
isolated from different matrices in different plants, (2) CT-RMAC, n.18, isolated
from skin samples in a single plant, and (3) EHLY agar, n.11, from different
matrices in different plants. All strains were confirmed by phenotypic identification
as
E. coli
, but none of the isolates were positive for the O157 serotype by the
in vitro
agglutination test. Based on the multiplex PCR results, the isolates (Fig.
27.1
)
belonged to three groups (Tables
27.2a
and
27.2b
): (1) VTEC, 43.4% (n.23), with
Table 27.1 VTEC
prevalence (%) as determined
by a PCR screening method in
relation to the matrix and
animal age
Matrix
Skin
Carcass
Mucosa
Feces
Sheep
13.7
9.5
6.3
-
Lamb
5.3
5.2
4.2
-
Total
18.9
14.7
10.5
-
