Biology Reference
In-Depth Information
7.2.3 RS-PCR Genotyping
For RS-PCR genotyping, the method described by Fournier et al. (
2008
) was used.
PCR products were analyzed using an Agilent 2100 Bioanalyzer with a DNA 7500
LabChip kit (Agilent Technologies, Palo Alto, CA). For interpretation of the
results, two patterns were considered different if two or more peaks of the electro-
pherogram differed in size.
7.3 Results
7.3.1 Virulence-Factors
All strains were positive for coagulase (
coa
), termonuclease (
nuc
), and a membrane
protein associated with antibiotic resistance (
fmt
B) gene, and were negative for
genes involved in host-cell invasion (
eta
,
etb
, and
sak
). In the analysis of coagulase
(
coa
) and protein A, the X region (
spa
) genes produced different-sized amplicons of
560 bp (6.6%), 640 bp (33.3%), 730 bp (36.6%), and 850 bp (23.5%), and of 150 bp
(20%), 180 bp (30%), 250 bp (3.5%), 270 bp (13.3%), 290 bp (16.6%), and 320 bp
(16.6%), respectively, which allowed for grouping of the isolates in clusters based
on the fragments obtained. Among the strains, 11 (36.6%) were enterotoxigenic and
coded for enterotoxins A, C, D, G, H, I, and J, with a prevalence of strains harboring
the
seg
and
seh
genes and
sed
and
sej
genes (10%). Enterotoxins B, E, and L were
never detected in the isolates. For the
luk
E (leukotoxin E) gene, 63.3% of the strains
were positive, while 80% and 76.6% of the strains were positive for
cna
and
clumping factor (
clf
A), respectively.
Half of the samples analyzed were MRSA (
mec
A positive) and one strain
possessed the
tsst
gene that encodes a toxin responsible for toxic shock syndrome.
Similarly, only one strain had the Panton-Valentine leukocidin (
Luk
S-
Luk
F/PV)
gene. Eighteen out of the 30 (60%) strains possessed a gene coding for a new
leukotoxin (
Luk
E-
Luk
D) and only three of these were positive for
Luk
M-
Luk
F/PV,
which have been identified in strains isolated from bovine milk (Jarraud et al.
2002
). Of the isolated strains, 3.3% and 6.6% were positive for
scn
and
chp
,
respectively. These mobile elements are usually present in strains involved in
human infections (Sung et al.
2008
).
7.3.2 Genotyping
Analysis of the 16S-23S rRNA from 30 strains revealed 16 different profiles
(Fig.
7.1
). Of these profiles, four strains originating from three herds located in
Piacenza, Como, and Sondrio had the same RS-PCR profile as the Swiss
