Prevalence of Salmonella spp., Shigella spp., and intestinal parasites among food handlers working in University of Gondar student’s cafeteria, Northwest Ethiopia

Background Food-borne infections continue to be a major public health problem at the international level. The issue becomes more serious in developing countries like Ethiopia. Objective This study aimed to examine the prevalence of Salmonella and Shigella species and intestinal parasites, as well as antimicrobial resistance patterns and associated factors among food handlers at the University of Gondar cafeteria in northwest Ethiopia. Methods An institutional-based cross-sectional study was conducted from February to June 2021 in the University of Gondar cafeterias. Data related to the socio-demographic characteristics and hygienic practices of study participants were collected using structured questionnaires. A total of 290 stool samples were collected from food handlers. Culture and conventional biochemical tests were used to isolate the Salmonella and the Shigella species. Wet mount, Formol-ether concentration, and Kato Katz techniques were applied to identify intestinal parasites. Additionally, drug susceptibility tests were performed using the disk diffusion method. Statistical analysis was done using SPSS version 26. Results Of 290 food handlers’ stool samples analyzed, Twenty-seven 27 (9.3%) were positive for both Salmonella and Shigella species. The prevalence of Salmonella and Shigella species was 16 (5.5%) and 11 (3.8%), respectively. Most of the isolated pathogens were resistant to tetracycline 19 (70.4%), and trimethoprim/sulphamethoxazole 19 (70.4%). The overall rate of multi-drug resistant Shigella and Salmonella isolate was 59.3%. Besides, Fifty-seven 57 (19.7%) of the participants were positive for one or more intestinal parasites. The most prevalent intestinal Parasitosis was E. histolytica/dispar 22 (7.6%), followed by G. lamblia 13 (4.5%), and Ascaris lumbricoides 11 (3.8) not washing hands after using the toilet (AOR: 4.42, 95% CI: 1.57, 10.56), and consuming unpasteurized milk (AOR: 3.14, 95% CI: 1.65, 3.96), were factors significantly associated with the prevalence of Salmonella, and Shigella infection. Similarly, not washing hands after using the toilet (AOR: 2.19, 95% CI: 1.0, 1.4), and consuming unpasteurized milk (AOR: 10.4, 95% CI: 3.8, 28.8), were factors significantly associated with the prevalence of intestinal parasites infection. Conclusion The prevalence of intestinal parasites, Salmonella, and Shigella species was high. Therefore, it is imperative to implement a public health policy that includes ongoing microbiological surveillance.


Introduction
Food-borne diseases (FBD) refer to illnesses caused by consuming pathogenic microorganisms, such as bacteria, fungus, viruses, and parasites, or their toxins in the case of bacteria and fungi (1)(2)(3).Additionally, FBD is caused by unprotected food handling and processing, and poor Sanitary conditions (4).It has recently become a worldwide and local health problem.Every year, two million deaths from food-borne illnesses are reported, affecting around one-third of the world's population (5).Across both developed and developing countries, it is a major public health concern (5).The World Health Organization (WHO) states that 600 million people worldwide become severely ill each year, with 420,000 dying as a result of food contamination.FBD affects an estimated 48 million people in the United States each year, resulting in 128,000 hospitalizations and 3,000 fatalities (6,7).Food-borne and waterborne infections induce diseases that are both short-term (such as nausea, vomiting, and diarrhea) and long-term (such as cancer, kidney or liver failure, tissue damage, brain disorders, and neurological abnormalities) and are also projected to cause approximately 700,000 deaths each year in Africa (8).
Gastrointestinal parasitic infections are prevalent in both developed and developing countries, with the largest frequency in less developed countries as a result of socioeconomic, demographic, and health-related behaviors, as well as inadequate personal hygiene and environmental sanitation (9).The most common way for intestinal parasitic infections to spread is through contaminated food and water, but they can be further transmitted from person to person through feco-oral contact (10).Nearly one-third of the populations in affluent countries suffer intestinal infections caused by parasites (11).Ascaris lumbricoides, Trichuris trichiura, hookworm, Entamoeba histolytica, and Giardia lamblia infect an estimated 1.2 billion, 795 million, 740 million, 500 million, and 2.8 million people worldwide, respectively (12,13).In Ethiopia, the burden of intestinal parasites (IPs) is extremely high.A third (26 million), a quarter (21 million), and one in every eight (11 million) Ethiopians are infected with Ascaris lumbricoides, Trichuris trichiura, and hookworm, respectively (14-16).As a result, Ethiopia has the second, third, and fourth largest burdens of ascariasis, hookworm, and trichuriasis, in sub-Saharan Africa, respectively (17).
Food-borne disease is also caused by enteric bacterial infections (EBIs), including those caused by the genus Shigella (which causes Shigellosis) and Salmonella (which causes Salmonellosis).They thus remain important public health concerns.Moreover, vaccinations do not produce immunity in young infants, and the development of antibiotic resistance makes clinical prevention and control of typhoid fever challenging, especially in Africa (18).Every year, Salmonella species cause 93.8 million episodes of gastroenteritis worldwide, resulting in 155,000 fatalities.Of these instances, 80.3 million were thought to be food-borne (19).Shigella species are more common in temperate and tropical areas.Shigella species cause an estimated 80-165 million cases of disease and 600,000 deaths worldwide each year (20).Salmonella and Shigella species resistant to commonly prescribed antibiotics are a threat to children and the general community (21).Ethiopia has been reported to exhibit considerable rates of resistance for Shigella and Salmonella species to tetracycline (52.5, 82.4%), trimethoprim/sulphamethoxazole (37.5, 76.5%), and ampicillin (60, 47.1%), respectively (22).
Food handlers are those who work in the food preparation and serving industry.If they have bacterial or parasite diseases in their gastrointestinal tract and maintain poor personal hygiene, they may pose a major risk of spreading IPs and EBIs to the general public.Asymptomatic food handlers are known to play an essential role in the spread of infections and continue to pose a hazard to the public's health (23)(24)(25)(26).Previous research has shown that food prepared in large quantities by a variety of food handlers at higher education institutions is frequently prone to contamination by infectious microorganisms or asymptomatic carriers, which can cause outbreaks of food-borne diseases (24,25).Furthermore, due to insufficient laboratory infrastructure that impedes effective detection and antibiotic susceptibility testing, Shigella, and Salmonella species isolation in most African laboratories, including Ethiopia, continues to be difficult (27).As a result, there is a dearth of current information on Shigella, and Salmonella species, and their patterns of antibiotic susceptibility, as well as the prevalence of intestinal parasites among Ethiopian food handlers.Therefore, the purpose of this study was to evaluate the frequency of intestinal parasites, Shigella, and Salmonella species in the University of Gondar cafeteria, as well as the patterns of antimicrobial resistance of the isolates along with associated factors.175 from Bahir Dar and 740 km from Addis Ababa.Currently, the University of Gondar Hospital and Student Cafeteria serve meals to over 37,000 students and staff.

Study population, sample size, and sampling technique
The source populations were all food handlers at the University of Gondar Hospital and the student Cafeteria, handled food.The study populations were food handlers who worked in the cafeteria at the University of Gondar, were available at the time of data collection, and gave their consent.Food handlers who used antibiotics and/or anthelminthic drugs and were not have willingness to give stool samples were also excluded during sample collection.
The sample size was determined by using a single population proportion formula considering the following assumptions: Zα/2 = 1.96 for the standard scale of 95% level of confidence, level of precision (d) = 3%, p = expected prevalence don by Mama and Alemu (6.9%) (28).

Socio-demographic data collection
Using a standardized questionnaire, data regarding sociodemographic characteristics and associated factors such as gender, age, education level, marital status, service years, medical checkups, the practice of hand washing after the toilet, hand washing using soap, fingernail status, wearing hair cover, the practice of drinking unpasteurized milk, eating raw meat, and eating raw vegetables were collected.The questionnaires' validity was checked for accuracy and completeness by conducting a pretest at Wogera prison center.

Specimen collection, handling, and transportation
Using a labeled, clean, wide-mouth plastic container and a clean, wooden applicator stick, 3 g of fresh stool samples were collected from food handlers per the recommended procedures (28).Then the samples were subsequently preserved in Cary Blair media (for culture) and a 10% (V/V) formalin solution (for wet mount and parasite concentration) was used.The samples were then promptly transported to the laboratory using a sample carrier.

Parasite identification
To identify intestinal protozoa, each sample was immediately analyzed with normal saline (0.85% NaCl) and Lugol's iodine.A Formol-ether concentration method and Kato Katz methods were employed to enhance the detection of intestinal parasites (28).

Isolation and identification of Shigella and Salmonella species
Using the standardized technique, Salmonella, and Shigella species were characterized and isolated (29).To enrich the bacteria, one milliliter of a stool sample was mixed with nine milliliters of Selenite F broth after being transferred from the Cary Blair medium (Oxoid, Ltd.UK) and incubated at 37°C for 24 h.MacConkey agar (21) (Oxoid, Ltd., UK), deoxycholate agar (DCA), and xylose lysine deoxycholate (XLD) agar (Oxoid, Ltd., UK) were used to subculture an inoculum from selenite F broth.After 24 h of incubation at 37°C, the growth of Salmonella and Shigella species was differentiated by their colony characteristic appearance on DCA agar (Shigella species shows pale colonies in color, Salmonella species black center pale color colonies) and XLD agar (Shigella has red color colonies, Salmonella species red with a presence of black color center).Discreet pure colonies with a characteristic of Salmonella-and/or Shigella-like species were further inoculated into standard biochemical tests such as Triple Sugar Iron Agar (TSI) (Oxoid, Ltd., UK), Sulfide Indole Motility Agar (SIM) (Oxoid, Ltd., UK), Urea Agar (Oxoid, Ltd., UK), Simmons Citrate Agar (Oxoid, Ltd., UK), and Lysine iron Agar (Oxoid, Ltd., UK) for species identification.Subsequently, according to the established technique, the results of the biochemical test results consistent with Salmonella and/or Shigella species were reported (Figure 1) (30, 31).Then discreet pure colonies presence or absence of black centered on DCA or XLD were picked and suspended in sterile normal saline (0.85% NaCl) for an antimicrobial susceptibility test (AST).

Quality control
Before data collection, the questionnaire was pretested and the data collectors were given training to guarantee the accuracy of the data.The collected data was reviewed daily for consistency and accuracy.Participants were given instructions on how to appropriately collect samples.Additionally, we checked the culture media, antibiotic discs, and other reagents' expiration dates ahead of time.By inoculating well-known strains E. coli American type culture collection (ATCC) 25922, S. typhimurium ATCC 14028 (35), and S. flexnerii ATCC 12022 (36), the culture media's quality was evaluated.Every laboratory procedure was carried out per standard operating protocols (37).

Statistical analysis
At first, all data were coded and reviewed for accuracy.After coding, the data were entered using EPI-info version 7. For further analysis, the data was transferred to SPSS version 26.For categorical

Ethical consideration
Ethical clearance was obtained from the Ethical Review Committee of the School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar.A support letter was obtained from the Department of Medical Microbiology at the University of Gondar with reference number SBLS/02/25/2021.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.For the proper parasitic and antimicrobial treatments, food handlers who tested positive for enteric infections (bacterial and parasite) were linked healthcare facility at the University of Gondar.

Socio-demographic characteristics of the study participants
In this study, 290 food handlers participated with a 100% response rate.The average age of the study participants was 32.1 years (standard deviation: ±8.7 years), with the majority of them 240 (82.8%) falling between the ages of 20 and 40.The majority 242 (76.9%) of these were females.Regarding educational status, 135 (46.6%) completed secondary school and above.Of the total respondents, 116 (40%), do not have a practice of hand washing after the toilet.181 (62.4%) of the total respondents had medical checks.Approximately over 71 (24.5%) of the participants in the study had a practice of consuming unpasteurized milk, while 59 (20.3%) and 148 (51%) regularly consumed raw meat and vegetables, respectively (Table 1).

Associated risk factors of Salmonella and Shigella, and intestinal parasite infection
After bivariate logistic regression analysis, the following factors were added to a multivariate logistic regression analysis: the practice of washing hands after using a toilet (p ≤ 0.001), the practice of consumption of unpasteurized milk (p ≤ 0.000), and consumption of raw vegetables (p ≤ 0.012).The practice of not washing hands after using the toilet (AOR: 4.42, 95% CI: 1.57, 10.56), and the practice of consuming unpasteurized milk (AOR: 3.14, 95% CI: 1.65, 3.96), were factors significantly associated with the prevalence of Salmonella, and Shigella infection (Table 2).After bivariate logistic regression analysis, the following factors were added to a multivariate logistic regression analysis: the practice of washing hands after using a toilet (p ≤ 0.007), the practice of consumption of unpasteurized milk (p ≤ 0.000), and consumption of raw vegetables (p ≤ 0.015).The practice of not washing hands after using the toilet (AOR: 2.19, 95% CI: 1.0, 1.4), and the practice of consuming unpasteurized milk (AOR: 10.4, 95% CI: 3.8, 28.8), were factors significantly associated with the prevalence of intestinal parasites infection (Table 3).

Antimicrobial resistance profiles of Salmonella and Shigella isolates
The following antibiotics were tested against Salmonella and Shigella isolates Amoxicillin/clavulanic acid, Cefotaxime, Ceftazidime, Ceftriaxone, and Cefoxitin, Gentamicin, Meropenem, and were used to assess the susceptibility profile of 16 Salmonella and 11 Shigella isolates and the results revealed that all isolates were susceptible to Meropenem 11 (100%).Of the Shigella isolates, 11 (100%) to tetracycline and Trimethoprim/Sulphamethoxazole were resistant, followed by 10 (91%) to Amoxicillin/clavulanic acid and 9 (82%) to gentamycin.Furthermore, the Salmonella isolates were resistant to tetracycline; Trimethoprim/Sulphamethoxazole and Amoxicillin/ clavulanic acid were 7 (46.7%)(Table 4).
In this study, drug resistance to at least three different classes of antimicrobial agents is referred to as multidrug resistance, the overall multidrug resistance was 16 (59.3%),and out of the 27 isolates, 8 (53.3%)Salmonella and 7 (63.6%)Shigella species were multidrugresistant isolates (Table 5).

Discussion
Poor food handling practices can cause food contamination, which in turn may result in food-borne diseases that could represent a risk to the health of the general population.Food handlers play a major role in the spread of pathogens (38)(39)(40).
In our study, 27 (9.3%) of Salmonella or Shigella species were detected in food handlers.This finding is similar to those of previous studies performed in Ethiopia in which the reported isolation rate was The magnitude of Salmonella and Shigella species in stool samples from food handlers at the University of Gondar cafeterias, Northwest Ethiopia, 2021.
In this study, the isolation rate of Shigella species was 3.8%; it can be a sign of inadequate food handler cleanliness and result in bacillary dysentery epidemics among the population of students.The results of the research are supported by the findings in Ethiopia, Arba Minch (3%) (42), Dilla (3.2%) (21), and Gondar (2.7-3.1%)(45,51).Nevertheless, less than the results of other research done in Nigeria (15.5%) (43).These might result from the food handlers' irregular training in food preparation, handling, and cleanliness procedures, as well as variations in study years.
In this investigation, E. histolytica/dispar was the main parasite found in 7.6% of the stool samples analyzed.This is consistent with earlier research that was done in Ethiopia, where this parasite was found most frequently (15,55).E. histolytica is one of the typical protozoan parasites that cause human gastroenteritis, along with Giardia lamblia 4.5% (56).It spreads mostly by fecal-oral contact, and in areas where poor hygiene standards are prevalent, contaminated hands play a significant part in its transmission (57,58).
In this study, Food handlers who did not have a hand-washing practice after the toilet were two times more likely to be positive for intestinal parasites and Shigella and Salmonella species compared to those who had a hand-washing habit after using the toilet.Food handlers who drank unpasteurized milk were 10 times more likely to be positive for intestinal parasites and Shigella and Salmonella species compared to those who drank pasteurized milk.Food handlers this means that most of the intestinal parasitosis, Salmonellosis, and Shigellosis were transmitted by lack of hand washing practice after toilet and drunken unpasteurized milk.This study is a consistent study with Ethiopia, Gondar (51), Bahir Dar (59), Motta (60), Dilla (21), and Mekelle (61).Poor hygienic environments, inadequate toilets, and a lack of readily available facilities for hand washing practices all contribute to the spread of Salmonella and Shigella species.The majority of the university's food handlers reported only washing their hands with water, and some of them admitted to skipping the step entirely after using the bathroom (62).In this study, regarding antimicrobial susceptibility test results, a high rate of isolate susceptibility was observed to a few of the antimicrobial agents which is comparable to the study conducted at Debre Markos University, Ethiopia (41).However, Shigella and Salmonella isolates showed a high resistance rate to tetracycline, Trimethoprim/Sulphamethoxazole, and Augmentin which is concordant with the previous report from Gondar Town Health Institution, Ethiopia (63).This may have been caused by the extensive and unnecessary use of antibiotics.In the current practice of Ethiopia, it is simple for the general people to walk into a pharmacy and purchase antibiotics off the market.The high resistance rates seen could be explained by this indiscriminate usage of antibiotics (64).
The Salmonella species were also resistant to many of the antibiotics in the present study: 46.7% to Tetracycline, Amoxicillin/ clavulanic acid, and Trimethoprim/Sulphamethoxazole.This is comparable with a study done in Ethiopia, Gondar (51), Debre Markos University (41), Tigrai (58), and Dilla (21), which indicated that antimicrobial resistance of Salmonella species is an increasing concern.These results suggested that Ethiopian antimicrobial resistance is still a problem that has to be addressed.Antibiotic resistance has emerged as a significant global public health problem, necessitating coordinated action (67).Antibiotic resistance is mostly caused by changes in bacterial genomes, inappropriate antibiotic use, poor drug regulation rules, incorrect drug prescriptions, and disobedience to prescriptions (68).In this study, Salmonella isolates were highly susceptible to Ceftriaxone which was comparable with previous reports in Ethiopia (21,69).The magnitude of MDR Shigella and Salmonella species in this study was 63.6 and 53.3%, respectively, which is comparable with the previous findings in Ethiopia (21,69) but lower than 100% resistance in Addis Ababa, Ethiopia (46).The high MDR rate of Salmonella and Shigella isolates for most of the antibiotics currently used could limit our antibiotic options for empirical therapy.

Limitations
Due to a lack of available materials, the Shigella and Salmonella isolates in our investigation could only be identified at the species level.Furthermore, due to financial constraints, it was not possible to differentiate between the Entamoeba complex species (E.dispar and E. histolytica) by using molecular methods.

Conclusion and recommendations
In this study, the overall prevalence of Salmonella species and Shigella species was high as compared to the previous reports.Salmonella species was highly resistant to Trimethoprim/ Sulphamethoxazole, amoxicillin-clavulanic acid, and Tetracycline, however highly susceptible to Meropenem, Ciprofloxacin, Cefoxitine, and Chloramphenicol.Shigella species also show a high resistance rate to Trimethoprim/Sulphamethoxazole, Tetracycline, amoxicillin-clavulanic acid, and Gentamicin, and showed the least resistance to Ceftazidime, and Cefotaxime.Among the total Salmonella and Shigella isolates, multidrug resistance was documented in 59.3% of all bacterial isolates.The overall prevalence of different intestinal parasites from stool samples was 19.65%.Among those, the most prevalent intestinal parasitosis was E. histolytica/dispar, followed by G. lamblia.Regarding risk factors, consumption of unpasteurized milk, and no hand washing practice after the toilet showed high significant correlation with intestinal parasites, Shigella, or salmonella infection.Through continuing health education and training programs, the level of knowledge, as well as the standard of conduct of food handlers at cafeteria facilities, must be improved.To improve the personal hygiene of the food handlers, it is also advised that the required facilities be provided, such as a clean toilet and a safe water supply.Additionally, to prevent microbial infections in food handlers, it is recommended that consumers drink pasteurized milk, and constant epidemiological surveillance is recommended.

FIGURE 1 Flow
FIGURE 1Flow chart used for identification of Salmonella and Shigella species.

FIGURE 2
FIGURE 2 An institutional-based cross-sectional study was carried out among food handlers working at the University of Gondar Hospital and Student Cafeteria, northwest, Ethiopia from February to June 2021.The University of Gondar is located in Gondar at a distance of Abbreviations: AST, Antimicrobial Susceptibility Test; ATCC, American type culture collection; CLSI, Clinical and Laboratory Standard Institute; DCA, deoxycholate agar; EBIs, enteric bacterial infections; IPs, intestinal parasites; MAC, MacConkey agar; MDR, Multidrug resistance; XLD, xylose lysine deoxycholate.
, frequency distributions and percentages were calculated.The socio-demographic factors and the prevalence of Salmonella, Shigella, or intestinal parasites among the study participants were analyzed using Bivariable logistic regression.To determine factors that are statistically significantly associated with the presence of Salmonella, Shigella, or intestinal parasite infections, variables with p ≤ 0.2 in the Bivariable logistic regression analysis have proceeded to the multivariable logistic regression analysis.A p-value of <0.05 was considered statistically significant at a 95% confidence level. variables

TABLE 1
Socio-demographic characteristics of the study participants at the University of Gondar Cafeterias, Northwest Ethiopia, 2021.

TABLE 2
Associated risk factors of Salmonella and Shigella infection among the study participants at the University of Gondar Cafeterias, Northwest Ethiopia, 2021.

TABLE 3
Associated risk factors of intestinal parasite infection among the study participants at the University of Gondar Cafeterias, Northwest Ethiopia, 2021.
*p less than 0.05; COR, crude odds ratio; AOR, adjusted odds ratio; CI, confidence interval; N, the number of study participant.

TABLE 4
Antimicrobial resistance profile of Salmonella and Shigella isolates among study participants at the University of Gondar Cafeterias, Northwest Ethiopia, 2021.

TABLE 5
The magnitude of Multidrug-resistant Salmonella and Shigella isolates among study participants at the University of Gondar Cafeterias, Northwest Ethiopia, 2021.