MEDICINSKI GLASNIK

samples of poultry leg skin) from 53 different
markets in the central zone of the Zenica city
were examined for the presence of C. jejuni and
C. coli during thirteen sampling visits between
May and October 2002. Sampling was done by
laboratory technicians with a permission of market
managers. All poultry samples found in the
markets which originated from different countries
were sampled. Poultry meat samples came
from 14 national (81 samples) and 7 international
chicken meat producers (66 samples) imported
from Croatia and Germany (20 samples from
each country), Slovenia (22 samples), Turkey
and Holland (2 samples from each country). For
the isolation of Campylobacter from poultry
meat, the standardized procedure recommended
by ISO 10272 was followed (9,18). Chicken liver
or skin from legs (5 g) were enriched in 45 mL of
selective Preston broth (Oxoid), containing 5%
of horse blood (SR 048C, Oxoid) and incubated
in a microaerophilic atmosphere for 18 hours at
42 °C (CampyGen, Oxoid). One loopful of enrichment
broth was streaked on modified Preston
medium (Oxoid) and incubated in a microaerophilic
atmosphere at 42°C for 3 days.
We also isolated 15 C. jejuni C. coli strains
from 23 cloacal samples of chicken collected
from the biggest local farm with conventional
housing, and production of 750 kg/month. Sampling
was done by farm staff with farm owner’s
permission. A questionnaire about the contents of
food and antibiotics given to poultry (duration of
breeding before slaughtering, indication for antibiotic
distribution), monthly production, a place
of one-day storage of chickens was filled by a
farm owner. The single sampling took place in
July 2001. Isolates were stored at -70°C in a medium
consisting of nutrient broth No. 2 (CM0271
Oxoid) (32 g), agar (1.2 g), glycerol (150 mL)
and distilled water (up to 1000 mL), supplemented
with two vials of Campylobacter growth supplement
(SR0232E, Oxoid, Basingstoke, United
Kingdom).
After original isolation, the isolates were
long-term stored at -80°C in Biotechnical Faculty
in Ljubljana (Slovenia) for further studies.
Uzunović-Kamberović et al Antimicrobial resistance C. jejuni and C. coli
C. jejuni and C. coli were identified using
standard microbiological methods and multiplex
PCR (5,19).
The disc diffusion method was performed to
test the resistance to eight antimicrobials used in
human and veterinary medicine (Oxoid): ampicillin
(10 µg); erythromycin (15 µg); gentamicin
(10 µg), tetracycline (30 µg), chloramphenicol
(30 µg) nalidixic acid (30 µg), ciprofloxacin (5
µg); nitrofurantoin (300 µg). The inocula were
adjusted to turbidity of a 0.5 McFarland standard
and plated on Mueller-Hinton agar (Oxoid,
Basingstoke, UK) supplemented with 5% sheep
blood. The plates were incubated at 37°C for 48
hrs in a microaerobic atmosphere. The applied
susceptibility criteria were in accordance with
CLSI (NCCLS) [20]. The following cutoff values
were used: ampicillin, ≤ 13 mm, erythromycin, ≤
13 mm, gentamicin, ≤ 12 mm, tetracycline, ≤ 14
mm, chloramphenicol, ≤ 12 mm, nalidixic acid,
≤ 13 mm, ciprofloxacin, ≤ 15 mm, nitrofurantoin,
≤ 14 mm.
C. jejuni ATCC 3356, E. coli ATCC 25922
and Staphylococcus aureus ATCC 25923 control
strains were used.
Erythromycin and ciprofloxacin susceptibility
were further determined by Etest (AB Biodisc,
Solna, Sweden) as described previously (21).
The MICs (minimal inhibitory concentration)
of erythromycin and ciprofloxacin (Sigma
Aldrich, Saint Louis, USA) in Campylobacter
isolates were determined using the broth microdilution
method as described previously (20). For
erythromycin the method was slightly modified:
two-fold serial dilutions of erythromycin were
used at the concentrations from 0.015 to 512 μg/
mL. The MICs lowest concentration where no
growth was observed was determined on the base
of fluorescent signal measured by Microplate
Reader (Tecan, Mannedorf/Zurich, Switzerland)
after adding CellTiter-Blue Reagent ® following
manufacturer’s instructions (Promega Corporation,
Madison, USA) to culture the media. Isolates
were considered resistant to erythromycin
when MIC ≥ 32 mg/L, and resistant to cipro-
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