Prevalence and Antimicrobial Resistance Pattern of Salmonella, Shigella, and Intestinal Parasites and Associated Factor among Food Handlers in Dilla University Student Cafeteria, Dilla, Ethiopia

Background Food-borne disease is mostly caused by unsafe food handling and processing as well as poor hygienic practice. Recently, it is a worldwide and local burden to the human health. It is estimated that about one-third of the world population is affected by food-borne diseases annually and become a global public health problem. Hence, this study aimed to determine the prevalence, antimicrobial susceptibility patterns, and associated risk factors of Shigella, Salmonella, and intestinal parasites among food handlers in Dilla University, Southern Ethiopia. Methods An institutional-based cross-sectional study was conducted from November to September 2018/2019. A structured questionnaire was used for the collection of data on sociodemographic characteristics. Parasite and bacterial identification, as well as susceptibility testing, was done using standard parasitological and bacteriological procedures. Results Of the total 220 food handlers included in the study, 38.6%, 9.5%, and 3.2% were positive for intestinal parasites, Salmonella, and Shigella species, respectively. A. lumbricoides (11.4%) was the predominant parasite isolated followed by E. histolytica (7.7%). From the total Salmonella isolates, serogroup D was the most frequent isolate and from the total Shigella species, Shigella flexneri was the predominant isolate. In this study, through irregular medical checkups, those who drunk unpasteurized milk and ate raw meat were significantly associated with intestinal parasites. Both Salmonella and Shigella species were highly resistant to ampicillin (81%) and amoxicillin-clavulanic acid (>70%). Salmonella isolates are highly sensitive to cefotaxime and ceftriaxone, while Shigella is highly sensitive to ciprofloxacin and norfloxacin. MDR was recorded in 71.4% of all bacterial isolates. Conclusion The presence of a high prevalence of intestinal parasites, Salmonella, and Shigella species that were resistant to the commonly prescribed drugs is a treat to the children and the community at large. Therefore, measures including health education, improvement of safe water supply, sanitation facilities, and continuous monitoring of microbiological and antimicrobial surveillance are crucial.


Introduction
Food-borne diseases are the most common disease mostly caused by unsafe food handling and processing as well as poor hygienic practice. Recently, it is a global and local burden to the community health. It is estimated that about one-third of the world population is affected by food-borne disease annually and two million deaths were reported each year. It is a public health problem in developed and developing countries [1]. e epidemiology of food-borne problems is complex and associated with the virulence factor of the organism and change of lifestyle, knowledge, poor hygienic practice, international travel, and migration and poverty [1,2].
Both developed and underdeveloped countries are affected by intestinal parasitic infections throughout the world. Nearly one-third of the populations in developed countries are suffering from intestinal parasitic infection.
is is around five times higher in developing countries [3]. In Ethiopia, intestinal parasitic infections are mainly associated with factors like poor socioeconomic conditions, poor hygiene and sanitation practices, unsafe and inadequate water supply, and environmental change [4]. e most common etiologic agents of intestinal parasitic infections in Ethiopia are Ascaris Lumbricoides, Entamoeba histolytica, Giardia lamblia, Trichuris trichiura, and Hookworm [5,6].
Gastroenteritis is the most common health problem throughout the world which is caused by Salmonella and Shigella species [7,8]. WHO reported about 16 million new cases and 600,000 deaths from typhoid fever each year throughout the world [8]. Diarrhea and dysentery are the most common life threatening diseases caused by Shigella strains called Shigella dysentery [9]. Due to the price and scarcity of laboratory facilities to perform accurate detection and antimicrobial susceptibility testing, the information relating to Salmonella, Shigella species, and their antimicrobial susceptibility patterns in Ethiopia is scarce. us, this study was aimed to determine the prevalence of Salmonella species, Shigella species, intestinal parasites, and their antimicrobial susceptibility patterns among food handlers working at Dilla University cafeterias.

Study
Design. An institutional-based cross-sectional study was carried out among a total of 220 food handlers working at Dilla University, Southern Ethiopia, from November 2018 to September 2019. It found at a distance of 85 km from Hawassa and 365 km far from Addis Ababa. It is found in kola agroecological zone with an altitude of 1400 km above sea level and annual temperature ranging from 22°C to 29°C.

Sample Size and Sampling Technique.
e sample size was calculated using a single population proportion formula by taking the prevalence of intestinal parasites (14.5%) conducted in Aksum, 5% margin of error (d � 0.05) and 95% confidence interval (z � 1.96). e initial sample size was 191, and by considering a 15% nonresponse rate, the final sample size was determined to be 220. To select representative participants, the final sample size was proportionally allocated to each stratum, and food handlers were selected using a systematic random sampling technique. Participants who reported to have never used any antimicrobial in the last 2 weeks and during the study period were included in the study.

Data Collection and Intestinal Parasite Identification.
Data related to sociodemographic characteristics and personal hygiene practices were collected via a face to face interview method. After interviewing, respondents were asked to give a fresh stool specimen in a sterile, a clean widemouthed plastic container by clean wooden applicator stick, which was transported to microbiology laboratory for analysis. Two stool samples were collected; one stool sample will be immediately emulsified using saline (0.85% NaCl) for parasitological examination of trophozoite, cyst, larva, and ova stage. Direct wet mount preparation in normal saline, iodine solution, and formol-ether concentration sedimentation techniques were used for parasites identification.

Stool Culture.
e isolation and characterization of Salmonella and Shigella species were performed based on the standard procedure [10]. Briefly, a mixture of a stool sample (1 mL) was transferred from the Cary Blair medium into a tube containing 9 mL of Selenite F broth (Oxoid, Ltd. UK) and incubated at 37°C for 24 hours to enrich the bacteria. An inoculum from Selenite F broth was subcultured on deoxycholate agar (DCA) and xylose lysine deoxycholate (XLD) agar (Oxoid, Ltd UK). After overnight incubation at 37°C the growth of Salmonella and Shigella was differentiated by their colony characteristic appearance on XLD agar (Shigella: red colonies, Salmonella red with a black center) and DCA (Shigella: pale colonies, Salmonella black center pale colonies). Pure colony with or without black centered on DCA or XLD were picked and suspended in sterile normal saline (0.85% NaCl) [11].
2.6. Statistical Analysis. Data were edited, cleaned, entered, and analyzed using the statistical package for social science (SPSS) version 22. Descriptive analysis such as frequency and mean was used. Initially, the association between each exposure and the presence of infection was assessed using multivariate logistic regression and chi-square test, and odds ratios were computed to measure the strength of the 2 International Journal of Microbiology association. P value of <0.05 was considered to indicate statistically significant differences.

Ethical Consideration.
Ethical clearance was obtained from the Ethical Review Committee of Dilla University medical and health Science College. Written informed consent was obtained from each study participant. Strict confidentiality was maintained during the interview process and anonymity was kept during data processing and report writing. Food handlers who have found to be positive for enteric pathogens (parasite and bacterial) were referred to their respective staff medical center for appropriate antiparasitic and antimicrobial treatments.

Sociodemographic Characteristics.
A total of 220 food handlers were included in this study. Of these, 90.9% were females. e majority of the study subjects were between the age group of 20 and 40 years (84.5%) with the mean age of 31.2 (standard deviation ± 8.8 years). Most of the study  International Journal of Microbiology participants had completed secondary school (61%). 56.4% of the study participants were married. Most of the study participants had served in the cafeteria for more than 5 years (77.3%). Only study participants with educational status of elementary and secondary schools and food handlers serving in the cafeteria for <5 years were significantly associated with intestinal parasites (P value <0.05) ( Table 1).

Food Preparation and Personal Hygiene Related Factors of Food Handlers.
Of the total respondents, 51.4% reported that they always perform periodic medical checkups. Similarly, 79.5% reported that they always use soap and water for hand washing after visiting toilet. e finding from observation also supported that personal hygiene was practiced by the food handlers. From the total, 86.8% of the study participants had drunk unpasteurized milk and only 42.3% eat raw meat. Only study participants who did not perform a periodical medical checkup, those who drink unpasteurized milk, and those who were eating raw meat were significantly associated with intestinal parasites (P value <0.007) ( Table 2).

Prevalence of Salmonellosis and Shigellosis.
e overall prevalence of Salmonella and Shigella species was 12.7%. Of these, Salmonella and Shigella species each account for 9.5% and 3.2%, respectively. From the total Salmonella species, serogroup D was predominantly isolated with a prevalence of 52.4%, followed by serogroups C (33.3%) and B (14.3%). From the total Shigella species by biochemical identification. Shigella flexneri was predominantly isolated with the prevalence of 57.1%, followed by Shigella boydii with the prevalence of 28.6%.

Antimicrobial Resistance Pattern of the Isolated Salmonella and Shigella species.
Antimicrobial susceptibility tests showed that Salmonella isolates were highly resistant to ampicillin (81%), and amoxicillin-clavulanic acid and chloramphenicol each accounting for (71.4%). More than 85% of Salmonella isolates were sensitive to cefotaxime and ceftriaxone. Among the Salmonella isolates, serogroup D showed a higher resistance rate to ampicillin (81.8%) and amoxicillin-clavulanic acid (81.8%) and serogroup C showed higher resistance to ampicillin (85.7%). All Shigella isolates showed 85.7% resistance to ampicillin, cefuroxime, and amoxicillin-clavulanic acid. More than 85% of Salmonella isolates were sensitive to ciprofloxacin and norfloxacin (Table 4).

Antibiogram Pattern of Multidrug Resistant Salmonella and Shigella species.
Among the total isolates, multidrug resistance (MDR) was recorded in 71.4%. In this study, 66.7% of Salmonella isolates and 85.7% of Shigella isolates were recorded as MDR. Among Salmonella species, serogroup D (72.7%) followed by serogroup B (66.7%) showed a high level of MDR, while among Shigella species, S. sonnei and S. boydii showed100% level of multidrug resistance (Table 5).

Factors Associated with Intestinal Parasitosis among Food
Handlers. Multivariable logistic regression results showed that food handlers who served less than 5 years in the cafeteria, food handlers who did not attend periodic medical checkup, food handlers who drink unpasteurized milk, and food handlers who ate raw meat have shown significant association (P < 0.05) ( Table 6).

Discussion
In this study, a total of 220 food handlers were included. e prevalence of parasitic infection reported in the current study was 38.6%.
is is consistent with studies done in Bahirdar [18], in Nigeria [19], and in India [20] with prevalence of 41.1%, 38.1%, and 29.3%, respectively; but it is higher than the studies conducted in Iran [21] (11.9%), in Sudan [22] (6.9%), and in Ghana [23] (21.6%). However, it is lower as compared to the findings reported in Mekele [24] (49.3%) and in Anatolia [25] (52.2%). e differences might be due to variation in years of study, sociodemographic characteristics, personal hygiene practice and environmental sanitation, safe water supply, health promotion practice, food hygiene and safety training, knowledge of transmission, and prevention of intestinal parasite differences.
In the current study, Ascaris lumbricoides (11.4%) was the predominant parasite identified followed by Entamoeba histolytica (7.7%) and Giardia lamblia (6.4%). Similar findings have been reported in previous studies done in Ethiopia [26][27][28][29]. Studies done in Aksum town [6] (5%), in Iran [21] (3.7%), and Saudi Arabia [30] (9%) reported Giardia lamblia as the predominant intestinal parasite, but the study conducted in Addis Ababa [29] (70.8%) reported Entamoeba histolytica as the predominant parasite. e high prevalence of those parasites might be due to low personal hygiene practice and the easy mode of transmission of the parasite which is usually found in unsafe food, water, soil, or contaminated surface with feces. e finding of this study showed that the odds of being positive for intestinal parasitic infection is higher among food handlers who did not attend periodic medical checkup compared to those who had attended periodic medical checkup.
is figure is inconsistent with a report from Aksum [6], Mekelle [24], and Jimma Town [31]. In the current study, Food handlers who drink pasteurized milk were 81% less likely to be positive for intestinal parasites compared to those who drink unpasteurized milk. Food handlers who did not eat raw meat were 97% less likely to be positive for intestinal parasites compared to those who ate raw meat. is is to mean that most of the intestinal parasitosis was transmitted by eating raw meat and drinking unpasteurized milk.
In this study, the prevalence of Salmonella species was 9.5% which is in line with the finding reported in Arbaminch [32] (6.9%) and in Sodo town [33] (8.8%) but higher than the study conducted in different area of Ethiopia [29,[34][35][36] with prevalence ranging from 1.6% to 5%. On the contrary to our study, higher findings were reported in Ethiopia [37] and Nigeria [19] with a prevalence of 13.5% and 31.5%,  respectively.
e differences may be due to the environmental condition, study area, and the laboratory method used for the bacterial identification.
e isolation rate of Shigella species in this study was 3.2%, which may indicate food handlers' low hygienic status and may lead to outbreaks of bacillary dysentery among the student population. is finding is consistent with the study conducted in Arba Minch University [32] (3%) and Gondar [45] (2.7-3.1%), however, lower than the findings of other studies conducted in Nigeria [19] (15.5%), but the present result was higher than the reports in Abeokuta [46] (0%). e discrepancy may be due to the difference in a technique of pathogens isolation, type of study participant, lifestyle, and sample size. e resistance rates for the isolated Salmonella species in this study were high for ampicillin, amoxicillin, and chloramphenicol (>71%). Our finding was comparable with previous studies conducted in Sodo town [33], Gondar [45], Haramaya [47], and Central Ethiopia [48]. Cefotaxime and ceftriaxone resistant Salmonella isolates were not revealed in previous studies in Ethiopia [48,49] in contrary to our study. is may indicate the emerging of ceftriaxone and cefotaxime resistance isolates over time. Shigella species were highly resistant to ampicillin, amoxicillin, and cefuroxime.
is was consistent with the study done in another region of Ethiopia [32,45,47,50]. e magnitude of MDR Salmonella and Shigella species in this study was 66.7% and 85.7%, respectively, which is comparable with the previous findings in Ethiopia [33] but lower than 100% resistance in Addis Ababa University [29]. e high MDR rate of Salmonella and Shigella isolates for most of the antibiotics currently used could limit our antibiotic option for empirical therapy. International Journal of Microbiology e current study indicates a higher prevalence of Salmonella and Shigella species among food handlers who had between 1 and 5 years' work experience (71.5%) compared to less than 1 year (10.2%) or greater than 5 years (18.3%). is is in line with a study done in Mekelle University [51] in which 60% of food handlers who served for less than 5 years were infected. However, our finding is lower than that of the study done in Arba Minch University [32], where 32.4% of food handlers who served for greater than 5 years were infected. Lack of regular medical checkups, food safety training, inadequate supervision, and a low level of literacy of food handlers might contribute to this difference.

Conclusion
In this study, 38.6% of stool specimens were positive for different intestinal parasites. Salmonella isolation rate was 9.5%, of which >71% were resistant to ampicillin, amoxicillin-clavulanic acid, and chloramphenicol and >85% sensitive to cefotaxime and ceftriaxone. Shigella isolation rate was 3.2%, of which >85% were resistant to ampicillin, cefuroxime, and amoxicillin-clavulanic acid and showed the least resistance to ciprofloxacin and norfloxacin. Serogroup D among Salmonella species, Shigella flexneri among Shigella species, and A. lumbricoides among intestinal parasites were the predominant isolates. Among the total isolates, multidrug resistance was recorded in 71.4% of all bacterial isolates. Of the checked risk factors, irregular medical checkups, drinking unpasteurized milk, and eating raw meat were significantly associated with pathogens infection. erefore, constant epidemiological surveillance, improvement of personal hygiene, and drinking pasteurized milk and eating cooked meat are recommended to control pathogens infection in food handlers.

Abbreviations
AOR: Adjusted odds ratio AST: Antimicrobial susceptibility testing ATCC: American type culture collection CI: Confidence interval CLSI: Clinical and laboratory standards institute COR: Adjusted odds ratio MDR: Multidrug resistance SPSS: Statistical Package for Social Sciences TSB: Tryptone soya broth XLD: Xylose lysine deoxycholate agar WHO: World Health Organization.

Data Availability
All the datasets used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
Ethical clearance was obtained from the Research and Ethical Review Committee and approved by the Institutional Review Board, Dilla University. Permission was also obtained from Dilla University Human Resource Directorate.

Consent
Consent was obtained from each participant or guardian as incorporated in the "participant information sheet" before inclusion in this study. Written informed consent and assent were obtained from the study subjects before data collection.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Kuma Diriba designed the study, collected data, performed the experiments, analyzed the data, and prepared a draft and final write-up of the manuscript. Mr. Ephrem Awulachew participated in the conception and design of the study, performed the experiments and preparation of the draft, and final write-up of the manuscript. Mr. Zemach Ashuro collected data and participated in data analysis and interpretation. All authors reviewed and approved the final manuscript.