Samples: In this study, a total of 100 Turkish sausages samples that received heat
treatment during production were analyzed between April and June 2013 in Kayseri in Turkey. The sausage samples were purchased from
different retail markets periodically. The samples were
immediately transported to the laboratory in a cool box
and analysed within 1-2 h.
Reference Strain: Salmonella typhimurium (ATCC
13311) reference strain was used as positive control for
the isolation of Salmonella spp. The reference strain was
provided by Department of Microbiology, Faculty of
Medicine, University of Erciyes, Kayseri, Turkey.
Isolation of Salmonella spp.: For the isolation and
the characterization of Salmonella spp. from sausage
samples, the method proposed by ISO 6579 with minor
modifications was used12. In brief, 25 g sausage
samples were added to 225 mL volumes of buffer
peptone water (BPW CM1049, Oxoid, Basingstoke, UK).
The samples were homogenised for 2 min and incubated
for 24 h at 37 ºC. Then, dropping of 0.1 mL of preenrichment
aliquots were inoculated into tubes
containing 10 mL Rappaport Vassiliadis (RV) broth and
incubated for 48 h at 42 °C. From each of the RV
broths were inoculated onto Xylose Lysine Deoxycholate
(XLD) agar plates and incubated for 18-24 h at 37 °C.
Up to two suspect colonies with typical Salmonella
morphology were tested biochemically. Serological
tests were carried out using specific Salmonella O and
H agglutinating antisera (Difco 2537-47).
DNA Extraction and PCR Amplification: Total
genomic DNA was extracted from strains by using a
commercial AxyPrep™ Bacterial Genomic DNA
extraction kit (Axygen, Bioscience, USA) as described by
the manufacturer. The species specific primers were
used for the detection of the 23S rRNA gene as
described by Aabo et al.13. (ST11: 5'AGC CAA CCA
TTG CTA AAT TGG CGC A3′ and ST15: 5′GGT AGA
AAT TCC CAG CGG GTA CTG 3′). PCR was performed
in a reaction mixture of 50 μL final volume containing 5
μL template DNA, 5 μL 10XPCR buffer (Vivantis), 1.5 U
Taq polymerase (Vivantis), 500 μM dNTP Mix (Vivantis),
3 mM MgCl2 (Vivantis) and 25 pmol of each primer. PCR
amplification was carried out with an initial denaturation
of 95 °C for 1 min followed by 30 cycles, each consisting
of 94 °C for 15 s, 57 °C for 15 s and 72 °C for 30 s. The
final extension cycle was consisted of 8 min at 72 °C
(Techne TC-512). All amplification products were
determined by agarose gel (1.5%) electrophoresis at 100
V for 45 min (EC250-90, Thermo, USA). The gels were stained with ethidium bromide and visualized under a UV
transilluminator (Vilber Lourmat, Marne La Vallee,
France).
Determination of the Antimicrobial Sensitivity:
The antibacterial susceptibility testing of isolates to
ampicillin (AMP, 10 μg), cefazolin (KZ, 30 μg),
danofloxacin (DFX, 5μg), enrofloxacin (ENR, 5μg),
gentamicin (CN, 10 μg), nalidixic acid (NA, 30 μg),
neomycine(N, 10 μg), oxytetracycline (T, 30 μg) and
trimethoprim-sulfamethoxazol (SXT, 23.7 μg-1.25 μg)
was performed by disc diffusion method14. The
antimicrobial discs were purchased from Oxoid (UK)
except for enrofloxacin and danofloxacin, which were
obtained from Bayer (Germany).
Antimicrobial susceptibility test was carried out using
the disc diffusion method described by Bauer et al.14.
Briefly, the isolates were grown on Blood Agar (Merck,
1.10886) at 37 °C for 24 h. Then, a suspension of each
organism adjusted to McFarland 0.5 by using
physiological saline. The suspensions were spread onto
Mueller Hinton Agar (Merck, 1.05437). Antibiotic discs
were placed onto the agar and incubated at 37 °C for 24
h aerobically. After 24 h of incubation, the diameter of the
inhibition zones were measured with callipers and the
results were interpreted according to the CLSI standarts15.