Biology Reference
In-Depth Information
fraudulent and illegal products coming from China is extremely long: among them,
the retail of imported ichthyic species (
Protosalanx
spp. and
Neosalanx
spp.),
named “ice fish,” for morphologically similar species with a greater commercial
value, such as “bianchetto” (juvenile
S. pilchardus
) and “rossetto” (adult
A. minuta
), is recurring. Other factors that may considerably influence the quality
of the product, which is perishable, are the scarce information on fishing areas
(sweet and brackish water) and procedures, water microbial quality, freezing speed,
transport duration, and cold-chain maintenance. It is therefore appropriate to
perform a microbial characterization of these products, including qualitative path-
ogen identification (
Listeria monocytogenes
,
Salmonella enterica
, and
Vibrio
parahaemolyticus
) and the determination of the extent of spoilage bacteria
(mesophiles and psycrophiles) (Shin et al.
2004
; Twedt
1989
).
25.2 Materials and Methods
The microbial characterization of 43 samples of fresh fish (PF; 23 belonging to
S. pilchardus
and 20 to
A. minuta
), coming from the Adriatic, Tyrrhenian, and
Ligurian seas, was carried out. Samples were found in supermarkets and fishery
markets in the provinces of Parma and Pisa from 15 February to 15 April. Twenty-
one samples of ice fish (PG) and relative defreezing waters (PG H
2
O) from the
sweet water of China and purchased at supermarkets in the above-mentioned
provinces were investigated. Regarding the hygienic assessment, mesophilic aero-
bic count determinations of fresh fish (ISO standard 4833:2003) and psycrophilic
aerobic count determination of thawed fish (ISO standard 17410:2001) and the
defreezing waters were carried out. The presence of
Escherichia coli
(most proba-
ble number, MPN) (ISO standard 7251:2005) was also evaluated to verify potential
fecal contamination, resulting from the fishing waters. Detection of the pathogens
S.
enterica
and
L. monocytogenes
was carried out according to the standards UNI EN
ISO 6579:2008 and UNI EN ISO 11290-1:2005 (Reg. EC 2073:2005). Isolation of
V. parahaemolyticus
, not yet considered in the Regulation, was determined
according to the standard ISO/TS 21872-1:2007. The isolated
Salmonella
spp.
strains were serologically and genotypically identified (by PCR-Simplex). After-
wards, phenotypic (Kirby Bauer method) and genotypic (PCR-Simplex) typing
were executed to detect the presence of plasmid genes and virulence chromosomes.
For this purpose, 17 antibiotics were tested: enrofloxacin (5
g), ciproploxacin
m
(5
g), streptomycin (10
g), colistin sulfate (10
g), gentamicin (10
g),
m
m
m
m
cefotaxime (10
g),
sulfamethoxazole/trimethoprin (25
g), nalidixic acid
m
m
(30
g), cephalothin (30
g), kanamycin (30
g), tetracycline (30
g), neomy-
m
m
m
m
cin (30
g), chloramphenicol (30
g), amoxicillin/clavulanic acid (30
g), ampicil-
m
m
m
lin (30
g).
From each overnight culture, genetic material was extracted using a chelating
resin (instaGene
g), ceftazidime (30
g), and sulfonamide compounds (300
m
m
m
Matrix, BIORAD). Twenty to 24
l of a Master Mix consisting
™
m
of sterile, distilled water (Fermentas), Taq Buffer 1
(Fermentas), 0.2 mM of each
