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174 Medicinski Glasnik, Volumen 6, Number 2, August 2009 INTRODUCTION Campylobacter species are one of the most common inducers of bacterial diarrhoea in humans worldwide (1). Campylobacters is considered a zoonotic disease, and occur in the intestine of domestic, production and wild animals. Transmission to humans occurs via food, drinking water and pets (2,3). The major route of infection in humans is thought to be the consumption of contaminated poultry meat, probably because of the high prevalence of contamination of chicken carcasses with Campylobacter and the frequency of poultry consumption (2, 4-7). Although most reports based on molecular typing have shown a major contribution of poultry to human Campylobacter infections, its epidemiology is still not completely defined (8,9). Erythromycin and fluoroquinolones are considered the drugs of choice for the treatment of gastrointestinal infections. An increase of antibiotic resistance of human Campylobacter isolates, especially to fluoroquinolones has been reported in many countries, but resistance to erythromycin and other antimicrobials has also been observed (10-12). As Campylobacter may be transferred from animals to humans, the possible development of antimicrobial resistance in Campylobacter spp., due to the use of antimicrobial agents in food animals, is a matter of concern (10,13). C. jejuni isolated from clinical infections is generally susceptible to erythromycin, whereas a much higher level of erythromycin resistance among C. coli isolates has been reported (10,14). Until recently, the fact that the majority of C. coli isolates were usually obtained from pigs, suggested that they were the most probable food source of human infections with C. coli (14). Since 1991 in Zenica-Doboj Canton, Bosnia and Herzegovina region, a significant proportion of C. coli in human infections has been reported as compared to other countries in both asymptomatic carriers and diarrhoeic patients comprising 36% and 26.5% of thermo tolerant Camyplobacter isolates (15,16). Moreover, C. coli isolates showed no difference to erythromycin-resistance as compared to C. jejuni isolates in this region (11). The majority of C. coli isolates analyzed in previous studies was obtained from pigs, suggesting that pigs were the most probable source of human infections with C. coli, rather than chicken and cattle (17). If pigs were the source of human C. coli infections, it would be surprising given that the ethnic structure of the Zenica-Doboj Canton population has changed due to mass migration during the wartime, with Muslims accounting for 82.3% of the post-war population (Statistical yearbook of R/F B&H, Sarajevo, 2000, 2004). For Muslims the consumption of pork is almost nonexistent and for that reason in the Zenica city there was no pork available in the markets before 2004. This suggested that the primary source of C. coli infections might be other than pigs. The aim of this study was the comparison of antimicrobial resistance among C. jejuni and C. coli isolated from human infections, retail poultry meat and poultry in Zenica-Doboj Canton, Bosnia and Herzegovina. MATERIALS AND METHODS The Laboratory for Sanitary and Clinical Microbiology of the Cantonal Public Health Institute in Zenica serves a total population of 331,229 inhabitants in Zenica-Doboj Canton: 149,053 in the urban area and 182,176 in the rural area. During the entire year of 2002, stool specimens were received from 2,491 consecutive outpatients with sporadic diarrhoea. There were 1,557 specimens taken from children (0-6 years of age), 331 from elementary school students (7-14 years of age), 204 from high school students (15-19 years of age), 311 and 88 from adults (20-64 and >64 years of age, respectively). The samples were cultured on modified Preston medium (OXOID, Basingstoke, United Kingdom) and incubated in a microaerophilic atmosphere (CampyGen, OX- OID) at 42 °C for 48 h. A total of 147 samples of retail poultry meat products (25 samples of chicken liver and 122
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- 175
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