Study area
The study was carried out on food handlers (those participated in food preparation, dispatch, store and related services) of University of Gondar students’ dining rooms in all campuses. Laboratory investigation was done in the Veterinary Public Health and Microbiology Laboratory, University of Gondar. The University has a latitude and longitude of 12°36’N37°28’E with an elevation of 2133 meters above sea level and located 730 km north of Addis Ababa [8]. University of Gondar currently enrolled more than 26,000 students out of which more than half of them are getting dining services in the dining rooms included in this study. A total of 664 food handlers are working in these students dining rooms.
Sample size determination and sampling procedure
The sample size was determined by using a single population proportion formula [9] considering the following assumptions: Zα/2 = 1.96 for the standard scale of 95% level of confidence, level of precision = 5%, P = 0.5
The total sample size was 423 with 10% non response rate included.
Sampling and data collection procedure
Simple random sampling using lottery method was used to select the study subjects. Complete list of food handlers was obtained from human resource management of University of Gondar. Socio-demographic data was gathered by using structured pretested questionnaire and face to face interview. Stool specimens were collected from food handlers with a suitable labeled wide-mouthed plastic container and clean wooden applicator stick. Specimens were immediately transported to laboratory using ice box.
Bacteriological examination
Bacteriological examination was carried out based on standard procedures previously described [10]. Briefly: twenty five grams of stool sample was homogenized in 225 ml of buffered peptone water (BPW, Oxoid, England) within a sterile stomacher bag. The homogenate was mixed well using a laboratory blender (Stomacher 400, Seward, England) for 1 minute and incubated for 24 hours at 37°C. Then, 1 ml, 1 ml and 0.1 ml aliquot of the enrichment broths was transferred aseptically into 10 ml of Selenite Cystine (SC), 10 ml of Tetrathionate (T) and 10 ml of Rappaport-Vassilliadis (RV) broth, and incubated for 24 hours at 37°C, 37°C and 42°C, respectively. Following incubation, a loopful of each culture was streaked onto Brilliant Green Agar (BGA, Oxoid, England) and Xylose Lysine Deoxycholate (XLD, Oxoid, England) agar plates and incubated at 37°C for 24 to 48 hours. The plates (BGA and XLD) were examined for the presence of characteristics Salmonella colonies. A single positive colony showing red color with a black center on XLD and red color on BGA agars were subjected for biochemical tests for confirmation.
Biochemical tests
Identification of Salmonella was performed by subjecting presumptive colonies onto Triple Sugar Iron (TSI) agar, Lysine Iron agar, Methyl Red (MR) broth, Voges-Proskauer (VP) broth, Urea broth, Indole test, and Citrate utilization tests and incubated for 24 to 48 hours at 37°C. Colonies producing an alkaline slant with acid butt and hydrogen sulfide production on TSI, positive for lysine, negative for urea hydrolysis, negative for Indole test, negative for VP, positive for citrate utilization and positive for MR test were considered to be Salmonella[10]. Finally, all of the confirmed Salmonella isolates were examined for antimicrobial susceptibility.
Antimicrobial susceptibility test
All isolates were tested by Kirby-Bauer disk diffusion method using guidelines established by the Clinical and Laboratory Standards Institute (CLSI) [11]. In brief, by taking pure isolated colonies, bacterial suspension in test tubes was adjusted and compared to 0.5McFarland turbidity standards. The diluted bacterial suspension was then transferred to Mueller-Hinton agar plate using a sterile cotton swab and the plate was seeded uniformly by rubbing the swab against the entire agar surface followed by 24 hours incubation. Antibiotic impregnated discs were then applied to the surface of the inoculated plates using sterile forceps. The plates were then incubated aerobically at 37°C for 24 hours. E. coli (ATCC 25922), which was susceptible to all tested drugs, was used for quality control. A total of 9 selected antibiotic disks (Oxoid, UK) including amoxicillin (AML) 2 μg, ampicillin (AMP) 10 μg, cephalothin (KF) 30 μg, ceftriaxone (CRO) 30 μg, gentamicin (CN) 10 μg, nalidixic Acid (NA) 30 μg, sulphamethoxazole-trimetoprime (SXT) 25 μg, teteracycline (TE) 30 μg and nitrofurantoin (F) 100 μg were applied. Finally, the zone of inhibition was measured including the disk diameter and the susceptible, intermediate and resistant categories were assigned on the basis of the critical points recommended by the CLSI.
Study variables
Prevalence of Salmonella and their antimicrobial susceptibility patterns were dependent variables whereas sex, age, educational background, service year, hand washing habit, periodical medical examination, and food hygiene training status were independent variables considered in the current study.
Data processing and analysis
Data were cleaned and entered using Epi-Info version 3.5.4. The data then was transferred and analyzed using SPSS version 21. Descriptive statistics such as percentage were applied to compute the data.
Ethical consideration
Ethical clearance was obtained from Institutional Review Board of Institute of Public Health, College of Medicine and Health Sciences, University of Gondar. Written informed consent was obtained from all study participants.