Study area and study period
The study was conducted from November 2012 to June 2013 at Gondar town public dinning places. Gondar is located northwest of Ethiopia at latitude and longitude of 12°36’N and 37°28’E. Its altitude is 2200 meters above sea level. The maximum and minimum temperatures of the area are 30.7°C and 12.3°C, respectively. The total human population of the town is estimated to be 206,987. Gondar town, as one of most visited historical place in Ethiopia, hosts large number of visitors including foreign tourists . According to the information obtained from Gondar town trade and industry office in 2013, there are about 273 licensed public dinning establishments including 80 hotels, 18 restaurants, 65 bar and restaurants, 20 pastry and 90 butchery shops.
Study design and sampling
A cross-sectional study was conducted to determine the prevalence and antimicrobial susceptibility profile of Listeria isolates from foods of animal-origin samples purchased from a randomly selected public dinning houses (cafeterias, hotels, restaurants, pastry and retail shops) in Gondar town. Study samples were collected using simple random sampling based on proportional allocation from a complete list of public dinning places of the town.
Since there was no previous study in the area, sample size was estimated by taking 50% expected prevalence with 95% confidence interval and 5% desired accuracy level . Accordingly a total of 384 food samples consisting of 50 raw and 50 pasteurized milk, 40 cheese, 65 cream cakes, 20 ice cream, 85 minced raw meat, 24 pizza and 50 fish foods were collected aseptically using sterile plastic bags and transported immediately using icebox to microbiology laboratory. Out of each sampling unit that consisted of 100 g, 25 g analytical units were subsequently removed for microbiological analysis.
Identification of listeria species
Half Fraser broth (AES LAB., Combourg, France) was used as a primary selective enrichment medium. Ferric ammonium citrate (Sigma, Steinheim, Germany) was added as a supplement. Then, 25gm food sample was mixed with 225 ml half Fraser broth in a stomacher bag and homogenised using a laboratory blender, stomacher-400™ (Seward Medical, London, UK) at a higher speed for two minutes and incubated at 30°C for 24 h. Similarly, 25 ml milk was sampled and pH adjusted to neutral and thoroughly mixed with 1:10 ratio to half Fraser broth and incubated at 30°C for 24 h. On the second day, about 0.1 ml of culture was transferred to a tube containing 10 ml of Fraser broth (containing acriflavin hydrochloride 0.025 g/l of distilled water and nalidixic acid 0.02 g/l of distilled water)(AES Lab., Combourg, France) and ferric ammonium citrate. The inoculated medium was incubated at 37° for 48 h. Then a loopful of inoculum was taken from isolated colonies and streaked onto pre-dried sterile plates of PALCAM (Polymixin Acriflavin Lithium Chloride Ceftazidime Aesculin Mannitol). Plates were examined for grey-green colonies with black background, typical for Listeria spp. Selected representative colonies were further identified to species level based on haemolytic patterns and biochemical analysis, following previously described protocol by Rorvik and his associates .
For confirmation, from each PALCAM agar plates (BD Diagnostic Systems, Heidelberg/Germany), five colonies presumed to be Listeria species were taken and streaked onto the surface of pre-dried plates of Tryptone Soya Yeast Extract Agar (TSYEA) (Detroit, USA) and incubated at 37°C for 24 h or until growth is satisfactory. Typical colonies (1 mm to 2 mm in diameter, convex, colourless and opaque), were used for further biochemical tests. Gram staining, motility, haemolysis, catalase, and CAMP tests were also performed. Rhamnose (AES Lab., Combourg, France), Xylose (AES Lab., Combourg, France) and Mannitol (Merck, Darmstadt, Germany) fermentation was evaluated and positive reactions (acid formation) were indicated by a yellow colour within 24 to 48 h [9,10].
Antimicrobial susceptibility testing
Antimicrobial susceptibility test was performed for L. monocytogenes isolates using Kirby Bauer disc diffusion technique . About 2–3 pure colonies were taken from TSYEA, suspended in Muller Hinton broth and incubated at 37°C for 1–2 h. Bacterial suspension was then adjusted to 0.5 McFarland turbidity standards and transferred to Mueller-Hinton agar plate using a sterile cotton swab. Plates were seeded uniformly by rubbing the swab against the entire agar surface. After the inoculums were dried, antibiotic impregnated disks were applied to the surface of the inoculated plates using disc dispenser. The plates were then incubated aerobically at 37°C for 24 h. Finally, zone of inhibition was measured including the disk diameter. The susceptible, intermediate and resistant categories were assigned on the basis of the critical points recommended by Clinical and Laboratory Standards Institute (CLSI)  and according to the manufacturer’s leaflet attached to the disks. The antimicrobials (Oxoid Ltd, Basingstoke, Hampshlre, England) tested were amoxicillin (AML 20 μg), sulfamethoxazole-trimethoprime (SXT 25 μg), cephalothin (KF 30 μg), chloramphenicol (CAF 30 μg), cloxacillin (OX 30 μg), penicillin (P 10 μg), tetracycline (TE 30 μg), vancomycin (VA 30 μg), and gentamicin (CN 10 μg). Selection of antimicrobials was based on availability and frequent use of these antimicrobials in the study area both in veterinary and human medicine. L. monocytogenes ATCC 19111 were used as quality control strain.
Data management and analysis
Data was entered and analyzed using the statistical software SPSS version 20.0. Descriptive statistics was used to describe the frequency of Listeria species from different food samples, antimicrobial susceptibility pattern and hygienic conditions.
Ethical clearance was obtained from the Ethical Review Board of Institute of Public Health, University of Gondar. Written consent was received from public dinning house owners participated in this study. Confidentiality of the information was maintained throughout the study.