Biology Reference
In-Depth Information
exfoliative exotoxins. Since the strain contagiousity seems to depend on virulence
gene patterns, these factors could partially explain the different abilities some
S. aureus
strains have to colonize different host species or spread in the herd.
As recently found in Italy (Benedetti et al.
2010
), the antibiotic resistance of
mec
A-
positive strains (methicillin-resistant
S. aureus
, MRSA) could be indirectly
considered an additional virulence factor. In recent years, new methods with high
discriminative powers (PFGE, spa-typing, and MLST) for
S. aureus
genotyping
have been developed. These techniques have been used mainly for epidemiological
purposes to differentiate between strains. In Switzerland, a recent
S. aureus
genotyping study used a relatively simple method based on the amplification of
the 16S-23S rRNA intergenic spacer (RS-PCR); this technique is suitable for
studies involving the analysis of numerous samples in a short time (Fournier
et al.
2008
). This assay made it possible to classify strains with high virulence
and diffusivity into specific genotypic profiles. The aim of this study was to
determine whether methicillin-resistant
S. aureus
(MRSA) or methicillin-sensitive
S. aureus
(MSSA) strains isolated from different Italian herds were included in the
genetic profiles and virulence patterns similar to those documented in Switzerland.
7.2 Materials and Methods
7.2.1 Samples
Thirty
S. aureus
strains isolated from bovine mastitic milk samples were collected
from 20 different northern Italian herds. DNA was extracted from 1 ml of culture
broth (about 10
8
-10
9
cfu/ml), following the instructions described in Cremonesi
et al. (
2006
).
7.2.2 Analysis of Genes Associated with Virulence Factors
DNA was amplified to verify the presence of virulence-associated genes using
primers and protocols described previously [
nuc
,
sea
,
sec
,
sed
,
seg
,
seh
,
sei
,
sej
,
sek
, and
sel
(Cremonesi et al.
2005
);
seb
and
see
(Pinto et al.
2005
; Monday and
Bohach
1999
);
coa
,
clf
A,
spa
,
tsst
,
eta
, and
etb
(Akineden et al.
2001
);
leuk
E
(Fournier et al.
2008
);
Luk
S-
Luk
F/PV and
mecA
(McClure et al.
2006
);
sak
,
fmtB
,
scn
, and
chp
(Sung et al.
2008
);
LuK
E-
Luk
D and
Luk
M (Jarraud et al.
2002
); and
cna
(Zecconi et al.
2006
)].
