Occurrence and antimicrobial susceptibility of 
Foodborne diseases (FBDs) are acute diseases due to consumption of food contaminated with sufficient doses of pathogenic microbes or toxins . According to the first estimate of the global burden of foodborne diseases by the World Health Organization in 2010, 600 million people experienced foodborne diseases, resulting in 420,000 deaths and a loss of 33 million healthy years globally . The World Bank has estimated that the economic cost of FBDs in low- and middle-income countries (LMICs) is $95.2 billion per year, and the annual cost of treating foodborne illnesses is estimated to be $15 billion . In the African region, where the FBDs burden is the highest, nearly 70% of the burden is due to diarrheal disease agents, particularly Salmonella enterica . Animal source foods, such as dairy products, eggs, fish and meat, are important sources of high-quality nutrients; however, they are also the leading points of exposure to foodborne pathogens . Dairy products are responsible for 20 disability-adjusted life years (DALYs) per 100,000 people in 2010 . This corresponds to approximately 4% of the global burden of foodborne disease and 12% of the animal source FBDs burden. A previous study  in Rwanda revealed 57,500 illnesses, 55 deaths and 3,870 DALYs, resulting in a $3.2 million cost-of-illness associated with the consumption of dairy products. Of these, 44% of the burden was attributed to drinking raw milk. Salmonella enterica is among the most important pathogens responsible for such burdens . The most frequent causes of foodborne illness are diarrheal disease agents . Staphylococcus aureus , Salmonella enterica , Campylobacter species, Listeria monocytogenes , and Escherichia coli are the major foodborne zoonotic bacterial pathogens that cause FBDs, and death associated with the consumption of contaminated animal products . Among all FBDs, diarrhea due to Salmonella enterica infections results in the highest burden, causing 4.07 million DALYs . Dairy products, including raw milk and soft unpasteurized cheese, have been identified as sources of Salmonella enterica . In Ethiopia, diarrhea was reported to be the second greatest contributor to the total burden of all disease types and the leading cause of premature death . The hygiene of milk containers, the environment in which milking occurs, the bedding in cow barns, and the hygiene of people engaged in milking determine milk quality and safety . In most developing countries, including Ethiopia, the prevailing poor food handling practices, lack of access to clean water and sanitation services, inadequate food safety laws, weak regulatory systems, and lack of financial resources to invest in safer equipment are identified as the main risk factors for the occurrence and associated burden of FBDs . Antimicrobial agents represent one of the main tools both in human and veterinary medicine to control and treat variety bacterial infectious diseases. However, during the past decades, the inappropriate use of antimicrobials as therapeutic or prophylactic agents in human and veterinary medicine, as well as the use of growth promoters in animal production  has resulted in the emergence of antimicrobial resistant Salmonella enterica that has no longer responded to antimicrobial therapy probably due to continuous antibiotic pressure selection . AMR rates in Salmonella enterica isolates ranged from, 4.66–87.74, to commonly prescribed antimicrobials . Food is an important vehicle for the transfer of AMR factor to intestinal tract of consumers very efficiently . The routine practice of giving antimicrobials to domestic livestock for prophylaxis and growth promotion is an important factor in the emergence antibiotic resistance bacteria in the food chain such as milk supply chain . Antimicrobial resistance has also been linked to the excessive use of antimicrobial drugs in food processing, in animals and humans; however, little attention has been given . FBDs pathogens can develop resistance to a number of antimicrobial classes, leading to significant public health implications. Multidrug-resistant (MDR) foodborne pathogens including Salmonella enterica infection can be difficult to treat, increasing the risk of severe illness, hospitalization, and even death . Antimicrobial-resistant Salmonella enterica have been isolated from different foods of animal origin including dairy products around the world, resulting in higher morbidity and mortality rates and higher overall treatment costs. This may represent a public health risk by transfer of resistant Salmonella enterica to humans through the consumption of contaminated food and food products . Salmonella enterica are resistant to multiple antibacterial classes such as sulphonamides, ampicillin, streptomycin, chloramphenicol and tetracyclines . However, the sources and transmission routes of Salmonella enterica in developing countries, such as in Ethiopia, are poorly understood due to the lack of coordinated national epidemiological surveillance systems . Moreover, Ethiopia has a number of regulations and strategies to address antimicrobial resistance (AMR), including the national action plans, governance structure, and legal frameworks; however, they are not well implemented . Prior studies have reported that AMR prevalence in Ethiopia especially in wolaita sodo is increasing at an alarming rate. Pathogens including Salmonella enterica causing diverse disease conditions showed 30–85% resistance to key antimicrobial agents described in the Ethiopian Standard Treatment Guideline (STG) . Few studies conducted previously in different regions of Ethiopia reported an occurrence of Salmonella enterica ranging from 6.5 to 20% in dairy products such as milk . This study was mainly conducted on milk samples collected at specific points along the supply chain. In addition, previous studies did not investigate the potential sources of contamination in the milking environment or assess the antimicrobial susceptibility profile of Salmonella enterica isolates throughout the entire milk supply chain, including milk, the milking environment, and the milk supply chain and milk consumers. Understanding the epidemiology of Salmonella enterica infections associated with dairy farming and dairy products is critically important for reducing public health risks. The present study focuses on investigating the occurrence and antimicrobial susceptibility of Salmonella enterica along the milk supply chain and in humans in Woliata Sodo, Ethiopia.   Study area The study was conducted in Woliata Sodo, southern Ethiopia (Fig. ), from January 2020 to February 2022. Wolaita Sodo is a town located in south-central Ethiopia, approximately 390 km south of Addis Ababa, the capital city of Ethiopia. It is a political and administrative center of the Wolaita Zone and Southern Ethiopia. It is situated at latitudes and longitudes of 6°54′N and 37°45′E and has an elevation between 1,600 and 2,100 m (5,200 and 6,900 feet) above sea level . The average annual rainfall in the area is 1,014 mm , and the mean annual temperature is 19.9°C, with monthly temperatures ranging from 17.7°C to 22.1°C . According to the Central Statistical Authority , the total population of the Woliata zone is 1,707,079; the population of Sodo town is 76,780, 40,495 of whom are male and 36,285 of whom are female. There are two major dairy production systems in the Wolaita zone: mixed crop livestock and urban dairy systems . The livestock population of the Woliata zone is estimated at 896, 032cattle, 240,315 sheep, 159,362 goats, 228 horses and 1, 019, 6 poultry . Out of 896, 032cattle population, 265,349  were female milking cows in wolaita zone of which 418 lactating cows were listed by Agricultural zonal office in wolaita sodo during the study period. The average daily milk production per cow in Ethiopia was 1.48 L. However, the milk yield per cow varies depending on the breed of the cow and the region . Study settings The study involved the collection of samples from cows, humans, dairy equipment, and farm environments. Lactating cows from Sodo Town dairy farms, with no recent medication history, were selected based on farm owner or manager information. Smallholder dairy farms in this study were defined as those that have less than 10 cows and dominantly hold Zebu local breeds, whereas commercial dairy farms were defined as those that contain more than 20 cows, dominantly have Zebu – holistin F crossbreeds, and keep the cows for commercial milking purposes. In Wolaita Sodo, there are more than 200 smallholders, and more than 26 commercial dairy farms were registered at the Agricultural zonal office at the time of sampling. Of which, 106 dairy farms (80 small holders and 26 commercial dairy farms) were selected for the study. Human subjects included outpatients with a history of diarrhea who visited Otona Comprehensive Referral Hospital and regularly consumed milk and dairy products. Dairy farm personnel directly involved in milking were also included, along with dairy farms that distribute milk directly to consumers, regardless of their management system. Study design, sample size and sampling techniques A cross-sectional study design was employed to generate the desired data. Lactating cows within the farm were selected by a simple random lottery technique from each dairy farm. All commercial dairy farms (26) and small holder dairy farms (80), which had at least one lactating cow during the study period, were included in the study. From smallholder dairy farms (80 lactating cows), one lactating cow from each farm was selected, while two lactating cows and three cows from one farm with more than 30 dairy cows were selected from commercial dairy farms (26 lactating cows), respectively. A total of 133 udder milk samples were collected from the 106 dairy farms. The lactating cows on the farm were selected using a simple random lottery method for the first cow and systematic random techniques for commercial dairy farms. In addition, at least two samples were collected from the milking environment at each selected farm to detect the contamination source of Salmonella enterica . The sample size for the collection of stools from outpatient diarrheic human cases was estimated based on a prevalence of 7.2% for non typhodial Salmonella , a 5% margin of error, and a 95% confidence level using the formula described by Thrusfield , which resulted in 103 individuals. Stool samples were collected purposively by selecting individuals with diarrhea and a history of consumption of raw milk and milk products, since diarrhea is one of the clinical signs of Salmonella enterica . Before sampling, the demography of the patient was taken using semi structured questionnaires. 50 milk collection centers and 50 milk retailers were also included in this study, and they were selected using simple random lottery sampling techniques. The collecting centers and retailers were selected purposefully based on the number of customers used and accessibility to the customers. The milk supply chain in the study area includes the chain where raw milk reaches consumers from dairy farms, such as milk collectors, vendors, local markets and milk retailers (Fig. ). The environmental samples in the present study include the samples from milking environments such as milkers hand swabs, surface teat swabs, farm floor swabs, and equipment used for milking and storing milk swabs. In general, a total of 644 samples were collected for the study, comprising bulk tank milk ( n = 106), pooled milk ( n = 133), milkers’ hand swabs ( n = 59), teat surfaces ( n = 51), farm floors ( n = 37), milk storage containers ( n = 55), collectors’ bulk milk ( n = 50), retailers’ bulk milk ( n = 50), and stool from diarrheic patients ( n = 103). The study samples were collected in three phases. In phase I, samples from dairy farms; in Phase II, samples from milk collection centers and milk retailers; and finally, in phase III, stool samples from Otona teaching and referral comprehensive hospitals were collected (Fig. ). Milk sample collection Approximately 30 ml of milk sample was collected from each milk source (udder, collection centers and retail shops) into a sterile screw-capped universal bottle. Pooled udder milk samples were collected from all functional teats of each cow after discarding the first few streams of milk. Prior to sample collection, the udder and teats were cleaned and dried, and each teat end was scrubbed gently with cotton swabs moistened with 70% ethyl alcohol . Pooled bulk tank milk samples were taken after milk from all cows was collected into a bucket by thoroughly agitating the container. Similarly, the milk samples were collected from bulk containers from milk collectors and retailers. Collection of swab samples Swab samples were collected from milking personnel’s hands, milk storage containers, the farm floor of the milking house, and the teat surface of the cows. The sterile cotton-tipped swabbing technique was implemented according to the protocols recommended for microbial surface sampling as well as the sampling of human biological traces . The swab tip was moved across the surface in a zigzag pattern, like a wavy line going back and forth. Then, the swab was swept perpendicularly for a second time on the surface in the first sweeping direction, and the swabs were immediately placed in a sterile screw-capped tube containing 10 ml of Buffered Peptone Water (BPW, CM 0509 Oxoid, UK), quickly shipped to Wolaita Sodo University, Veterinary Microbiology Laboratory, through maintaining the cold chain, and stored at 4°C . All the swab samples were taken before milking. Stool sample collection Stool samples were collected from diarrheic outpatients. The patients were selected for clinical examination based on their history of diarrhea and habits related to milk and milk product consumption. Each study participant was requested to submit 2–3 g of freshly voided stool in a clean, dry, and leak-proof disposable stool cup provided by a health professional (nurse) to the clinical laboratory. Then, swabbing of the stool was performed by laboratory technicians at the hospitals. Upon completion of the swabbing process, the wooden shaft of the swab was broken off, and the cotton swab was immersed in test tubes containing 10 ml of sterilized BPW. The swab samples were shaken for 30 s for uniform distribution before transportation. All the sample-containing bottles were clearly labeled or coded with data and sample sources using waterproof ink and transported within 12 h of collection using an ice box containing ice packs to the Veterinary Microbiology Laboratory of Woliata Sodo University and stored at 4 °C until processing. Isolation and identification of Salmonella enterica The isolation and identification of Salmonella enterica were conducted according to ISO 6579-1 . Briefly, 25 ml of milk sample was transferred into 225 ml of sterile buffered peptone water (Oxoid, CM 0509, UK) for pre-enrichment and mixed well in a stomacher bag, whereas in the case of swabs or stool samples, the samples containing buffered peptone water (Oxoid, CM 0509, UK) were directly incubated at 37°C for 18 h. Then, 0.1 ml and 1 ml aliquots were aseptically inoculated into 10 ml of sterile Rappaport Vassiliadis (RV) broth (Difico, MD211, USA) and Muller Kaufmann Tetrathionate (MKTTn) broth (HIMIDIA, M14961, India), respectively. The cultures in RV broth were incubated at 41.5°C for 24 h, and those in the MKTTn broth were incubated at 37 °C for 24 h. A loopful of culture from the RV and MKTTn broths was streaked onto Xylose Lysine Deoxycholate (XLD) Agar (HIMIDIA, M031B, India) and Hektoen enteric agar (HE) (HIMIDIA, GM467, India) and incubated aerobically at 37°C for 24 h. Pink colonies with or without black centers on XLD and blue‒green to blue colonies with or without black centers on HE agar were considered presumptive Salmonella colonies. The presumptive colonies were selected from the selective plates, sub-cultured on brain heart infusion (BHI agar, BBL™, MD21152, USA), incubated at 37°C for 24 h and then stored for further analysis. The isolates were confirmed using biochemical tests. Each colony was inoculated into triple sugar iron (TSI) agar, methyl red-Voges-Proskauer (MR-VP) broth, Simmons’ citrate agar, and lysine iron agar (LIA) and incubated at 37°C for 18–48 h. The results of the biochemical tests were interpreted as described by ISO 6579-1:2017 . Latex agglutination test A latex agglutination test was further performed on the presumptive Salmonella enterica isolates identified by biochemical tests. The test was performed according to the manufacturer’s protocol ( Salmonella Latex kit, Microgen Bio product). Briefly, one drop of saline was added to a well of the reaction card, a typical suspected colony was emulsified in a drop of saline, and one drop of test latex (Oxoid, R6248pw, UK) was added and mixed with a sterile mixing stick for two minutes. The agglutination was examined, and the observed signs of agglutination were considered positive for Salmonella enterica . Polymerase chain reaction (PCR) confirmation Serologically identified Salmonella enterica isolates were sub-cultured from the stored isolates on selective media and then onto brain heart infusion (BHI agar, BBL™, MD21152, USA) agar and incubated at 37 ± 2°C overnight. Pure single colonies were picked from BHI agar, inoculated in 100 µl of sterile nuclease-free molecular grade water (Ambion, USA) using a sterile disposable plastic loop and placed on a heated thermal block at 95°C for 10 min in micro centrifuge tubes for cell lysis. The tubes were centrifuged in a mini centrifuge at the highest speed (14,000X) for 5 min, and then,50 µl of the supernatant was transferred to a new tube and stored at −20°C until being used for PCR analysis . PCR was carried out using a Bio-Rad T100 thermo cycler in a 25 µl reaction volume. Each reaction contained 2.5 µl of a DNA template, 12 µl of Promega GoTaq Green Master mix (containing Taq polymerase, dNTPs, MgCl 2 , and reaction buffer), 0.81 µl of 10 µM primer Salm3, 0.87 µl of 10 µM primers Salm4 and 8.82 µl of nuclease-free water (DEPC, BP561-1L, UK). The thermal cycling protocol was standardized to set the PCR assay as an initial denaturation step at 95°C for 5 min followed by 35 cycles of amplification using a thermal cycler (BIO RAD T100™ Thermal Cycler, Singapore). Each cycle consisted of denaturation at 95°C for 90 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 90 seconds. The PCR ended with a final extension at 72°C for 7 min. A positive control ( Salmonella enterica ATTCC35664) and a negative control (nuclease-free water) were used in each PCR run . The DNA was used as a template for the amplification of the highly conserved region of the invA gene using the primers Salm3 (5´-GCTGCGCGCGAACGGCGAAG-3’) and Salm4 (5´-TCCCGGCAGAGTTCCCATT-3´), which amplify a 389 bp fragment of the conserved invA gene sequence of Salmonella enterica . The result of the PCR was included as a supplementary file. PCR amplicons were confirmed by gel electrophoresis using a 1.5% w/v agarose gel (Thermo Scientific, 17852) by mixing 1.5 g of agarose with 100 ml of 1x TAE buffer (BDH4616, USA) in a glass flask supplemented with 5 µl of red gel (5 mg/ml). DNA was separated at 120 volts for 40 min. The gel images were viewed and visualized with a Bio-Rad Gel Doc EZ Gel Documentation System using a UV trans-illumination system (UVP Bio Doc-It Imaging Systems, Analytik Jena). Bands of 389 base pairs (bps) were identified as Salmonella enterica . A 100-bp DNA ladder (product no N32315) was used as a molecular dimension marker to estimate the size of the products. Antimicrobial susceptibility test The antimicrobial susceptibility of the Salmonella enterica isolates was determined according to the Kirby-Bauer Disk Diffusion method on Muller Hinton agar (HIMEDIA, M173, India) . Pure colonies from brain heart infusion agar were removed with a disposable plastic loop, transferred to a tube containing 5 ml of brain heart infusion broth, and emulsified. The emulsified broth culture was incubated at 37°C overnight and compared with 0.5 McFarland turbidity standards. The cotton swabs were immersed in emulsified broth, the contents were smeared evenly on Muller Hinton agar plates, and the operation was performed under a laboratory safety cabinet (level II). The plates were incubated at room temperature for 15 min to allow drying. Antibiotic discs with known concentrations of antimicrobials were carefully placed on plates, and the plates were incubated for 24 h at 37°C. Following incubation, the diameters of the clear zones produced by antimicrobial inhibition of bacterial growth were measured to the nearest millimeter for each disc using a transparent straight-line ruler and then classified as resistant or susceptible according to the Clinical Laboratory Standards Institute . Fourteen antibiotics belonging to different antimicrobial classes were selected based on their clinical and veterinary relevance . The antibiotic agents (Oxoid, UK) used were amoxicillin (30 µg), ampicillin (10 µg), ceftazidime (30 µg), clindamycin (2 µg), ciprofloxacin (5 µg), Ceftriaxone (30 µg), Chloramphenicol (30 µg), doxycycline (30 µg), erythromycin (15 µg), norfloxacin (10 µg), streptomycin (10 µg), vancomycin (30 µg), trimethoprim (5 µg) and tetracycline (30 µg). The multidrug resistance (MDR) (resistance to three and more than three antimicrobial classes) characteristics of the isolates were identified by observing the resistance patterns of the isolates to the antibiotics . A total of 12 PCR confirmed Salmonella enterica positive isolates (3 from environmental, 2 from stool and 7 from milk supply chain) were used for the analysis. Data management and analysis Data entry and management were performed using Microsoft Office Excel 2010, ensuring that all entries were accurately recorded and saved. After thorough data screening to check for errors and completeness, data analysis was performed using STATA version 14 (StataCorp, 4905 Lakeway Drive, College Station, Texas 77845 USA). Descriptive statistics such as frequency and percentage were employed to summarize the data. Chi square(x 2 ) test was used to assess the association between the contamination of Salmonella enterica and independent variables. The multidrug resistance (MDR) characteristics of the isolates were identified by observing the resistance patterns of the isolates to the antibiotics .   The study was conducted in Woliata Sodo, southern Ethiopia (Fig. ), from January 2020 to February 2022. Wolaita Sodo is a town located in south-central Ethiopia, approximately 390 km south of Addis Ababa, the capital city of Ethiopia. It is a political and administrative center of the Wolaita Zone and Southern Ethiopia. It is situated at latitudes and longitudes of 6°54′N and 37°45′E and has an elevation between 1,600 and 2,100 m (5,200 and 6,900 feet) above sea level . The average annual rainfall in the area is 1,014 mm , and the mean annual temperature is 19.9°C, with monthly temperatures ranging from 17.7°C to 22.1°C . According to the Central Statistical Authority , the total population of the Woliata zone is 1,707,079; the population of Sodo town is 76,780, 40,495 of whom are male and 36,285 of whom are female. There are two major dairy production systems in the Wolaita zone: mixed crop livestock and urban dairy systems . The livestock population of the Woliata zone is estimated at 896, 032cattle, 240,315 sheep, 159,362 goats, 228 horses and 1, 019, 6 poultry . Out of 896, 032cattle population, 265,349  were female milking cows in wolaita zone of which 418 lactating cows were listed by Agricultural zonal office in wolaita sodo during the study period. The average daily milk production per cow in Ethiopia was 1.48 L. However, the milk yield per cow varies depending on the breed of the cow and the region .   The study involved the collection of samples from cows, humans, dairy equipment, and farm environments. Lactating cows from Sodo Town dairy farms, with no recent medication history, were selected based on farm owner or manager information. Smallholder dairy farms in this study were defined as those that have less than 10 cows and dominantly hold Zebu local breeds, whereas commercial dairy farms were defined as those that contain more than 20 cows, dominantly have Zebu – holistin F crossbreeds, and keep the cows for commercial milking purposes. In Wolaita Sodo, there are more than 200 smallholders, and more than 26 commercial dairy farms were registered at the Agricultural zonal office at the time of sampling. Of which, 106 dairy farms (80 small holders and 26 commercial dairy farms) were selected for the study. Human subjects included outpatients with a history of diarrhea who visited Otona Comprehensive Referral Hospital and regularly consumed milk and dairy products. Dairy farm personnel directly involved in milking were also included, along with dairy farms that distribute milk directly to consumers, regardless of their management system.   A cross-sectional study design was employed to generate the desired data. Lactating cows within the farm were selected by a simple random lottery technique from each dairy farm. All commercial dairy farms (26) and small holder dairy farms (80), which had at least one lactating cow during the study period, were included in the study. From smallholder dairy farms (80 lactating cows), one lactating cow from each farm was selected, while two lactating cows and three cows from one farm with more than 30 dairy cows were selected from commercial dairy farms (26 lactating cows), respectively. A total of 133 udder milk samples were collected from the 106 dairy farms. The lactating cows on the farm were selected using a simple random lottery method for the first cow and systematic random techniques for commercial dairy farms. In addition, at least two samples were collected from the milking environment at each selected farm to detect the contamination source of Salmonella enterica . The sample size for the collection of stools from outpatient diarrheic human cases was estimated based on a prevalence of 7.2% for non typhodial Salmonella , a 5% margin of error, and a 95% confidence level using the formula described by Thrusfield , which resulted in 103 individuals. Stool samples were collected purposively by selecting individuals with diarrhea and a history of consumption of raw milk and milk products, since diarrhea is one of the clinical signs of Salmonella enterica . Before sampling, the demography of the patient was taken using semi structured questionnaires. 50 milk collection centers and 50 milk retailers were also included in this study, and they were selected using simple random lottery sampling techniques. The collecting centers and retailers were selected purposefully based on the number of customers used and accessibility to the customers. The milk supply chain in the study area includes the chain where raw milk reaches consumers from dairy farms, such as milk collectors, vendors, local markets and milk retailers (Fig. ). The environmental samples in the present study include the samples from milking environments such as milkers hand swabs, surface teat swabs, farm floor swabs, and equipment used for milking and storing milk swabs. In general, a total of 644 samples were collected for the study, comprising bulk tank milk ( n = 106), pooled milk ( n = 133), milkers’ hand swabs ( n = 59), teat surfaces ( n = 51), farm floors ( n = 37), milk storage containers ( n = 55), collectors’ bulk milk ( n = 50), retailers’ bulk milk ( n = 50), and stool from diarrheic patients ( n = 103). The study samples were collected in three phases. In phase I, samples from dairy farms; in Phase II, samples from milk collection centers and milk retailers; and finally, in phase III, stool samples from Otona teaching and referral comprehensive hospitals were collected (Fig. ).   Approximately 30 ml of milk sample was collected from each milk source (udder, collection centers and retail shops) into a sterile screw-capped universal bottle. Pooled udder milk samples were collected from all functional teats of each cow after discarding the first few streams of milk. Prior to sample collection, the udder and teats were cleaned and dried, and each teat end was scrubbed gently with cotton swabs moistened with 70% ethyl alcohol . Pooled bulk tank milk samples were taken after milk from all cows was collected into a bucket by thoroughly agitating the container. Similarly, the milk samples were collected from bulk containers from milk collectors and retailers.   Swab samples were collected from milking personnel’s hands, milk storage containers, the farm floor of the milking house, and the teat surface of the cows. The sterile cotton-tipped swabbing technique was implemented according to the protocols recommended for microbial surface sampling as well as the sampling of human biological traces . The swab tip was moved across the surface in a zigzag pattern, like a wavy line going back and forth. Then, the swab was swept perpendicularly for a second time on the surface in the first sweeping direction, and the swabs were immediately placed in a sterile screw-capped tube containing 10 ml of Buffered Peptone Water (BPW, CM 0509 Oxoid, UK), quickly shipped to Wolaita Sodo University, Veterinary Microbiology Laboratory, through maintaining the cold chain, and stored at 4°C . All the swab samples were taken before milking.   Stool samples were collected from diarrheic outpatients. The patients were selected for clinical examination based on their history of diarrhea and habits related to milk and milk product consumption. Each study participant was requested to submit 2–3 g of freshly voided stool in a clean, dry, and leak-proof disposable stool cup provided by a health professional (nurse) to the clinical laboratory. Then, swabbing of the stool was performed by laboratory technicians at the hospitals. Upon completion of the swabbing process, the wooden shaft of the swab was broken off, and the cotton swab was immersed in test tubes containing 10 ml of sterilized BPW. The swab samples were shaken for 30 s for uniform distribution before transportation. All the sample-containing bottles were clearly labeled or coded with data and sample sources using waterproof ink and transported within 12 h of collection using an ice box containing ice packs to the Veterinary Microbiology Laboratory of Woliata Sodo University and stored at 4 °C until processing.   Salmonella enterica The isolation and identification of Salmonella enterica were conducted according to ISO 6579-1 . Briefly, 25 ml of milk sample was transferred into 225 ml of sterile buffered peptone water (Oxoid, CM 0509, UK) for pre-enrichment and mixed well in a stomacher bag, whereas in the case of swabs or stool samples, the samples containing buffered peptone water (Oxoid, CM 0509, UK) were directly incubated at 37°C for 18 h. Then, 0.1 ml and 1 ml aliquots were aseptically inoculated into 10 ml of sterile Rappaport Vassiliadis (RV) broth (Difico, MD211, USA) and Muller Kaufmann Tetrathionate (MKTTn) broth (HIMIDIA, M14961, India), respectively. The cultures in RV broth were incubated at 41.5°C for 24 h, and those in the MKTTn broth were incubated at 37 °C for 24 h. A loopful of culture from the RV and MKTTn broths was streaked onto Xylose Lysine Deoxycholate (XLD) Agar (HIMIDIA, M031B, India) and Hektoen enteric agar (HE) (HIMIDIA, GM467, India) and incubated aerobically at 37°C for 24 h. Pink colonies with or without black centers on XLD and blue‒green to blue colonies with or without black centers on HE agar were considered presumptive Salmonella colonies. The presumptive colonies were selected from the selective plates, sub-cultured on brain heart infusion (BHI agar, BBL™, MD21152, USA), incubated at 37°C for 24 h and then stored for further analysis. The isolates were confirmed using biochemical tests. Each colony was inoculated into triple sugar iron (TSI) agar, methyl red-Voges-Proskauer (MR-VP) broth, Simmons’ citrate agar, and lysine iron agar (LIA) and incubated at 37°C for 18–48 h. The results of the biochemical tests were interpreted as described by ISO 6579-1:2017 .   A latex agglutination test was further performed on the presumptive Salmonella enterica isolates identified by biochemical tests. The test was performed according to the manufacturer’s protocol ( Salmonella Latex kit, Microgen Bio product). Briefly, one drop of saline was added to a well of the reaction card, a typical suspected colony was emulsified in a drop of saline, and one drop of test latex (Oxoid, R6248pw, UK) was added and mixed with a sterile mixing stick for two minutes. The agglutination was examined, and the observed signs of agglutination were considered positive for Salmonella enterica .   Serologically identified Salmonella enterica isolates were sub-cultured from the stored isolates on selective media and then onto brain heart infusion (BHI agar, BBL™, MD21152, USA) agar and incubated at 37 ± 2°C overnight. Pure single colonies were picked from BHI agar, inoculated in 100 µl of sterile nuclease-free molecular grade water (Ambion, USA) using a sterile disposable plastic loop and placed on a heated thermal block at 95°C for 10 min in micro centrifuge tubes for cell lysis. The tubes were centrifuged in a mini centrifuge at the highest speed (14,000X) for 5 min, and then,50 µl of the supernatant was transferred to a new tube and stored at −20°C until being used for PCR analysis . PCR was carried out using a Bio-Rad T100 thermo cycler in a 25 µl reaction volume. Each reaction contained 2.5 µl of a DNA template, 12 µl of Promega GoTaq Green Master mix (containing Taq polymerase, dNTPs, MgCl 2 , and reaction buffer), 0.81 µl of 10 µM primer Salm3, 0.87 µl of 10 µM primers Salm4 and 8.82 µl of nuclease-free water (DEPC, BP561-1L, UK). The thermal cycling protocol was standardized to set the PCR assay as an initial denaturation step at 95°C for 5 min followed by 35 cycles of amplification using a thermal cycler (BIO RAD T100™ Thermal Cycler, Singapore). Each cycle consisted of denaturation at 95°C for 90 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 90 seconds. The PCR ended with a final extension at 72°C for 7 min. A positive control ( Salmonella enterica ATTCC35664) and a negative control (nuclease-free water) were used in each PCR run . The DNA was used as a template for the amplification of the highly conserved region of the invA gene using the primers Salm3 (5´-GCTGCGCGCGAACGGCGAAG-3’) and Salm4 (5´-TCCCGGCAGAGTTCCCATT-3´), which amplify a 389 bp fragment of the conserved invA gene sequence of Salmonella enterica . The result of the PCR was included as a supplementary file. PCR amplicons were confirmed by gel electrophoresis using a 1.5% w/v agarose gel (Thermo Scientific, 17852) by mixing 1.5 g of agarose with 100 ml of 1x TAE buffer (BDH4616, USA) in a glass flask supplemented with 5 µl of red gel (5 mg/ml). DNA was separated at 120 volts for 40 min. The gel images were viewed and visualized with a Bio-Rad Gel Doc EZ Gel Documentation System using a UV trans-illumination system (UVP Bio Doc-It Imaging Systems, Analytik Jena). Bands of 389 base pairs (bps) were identified as Salmonella enterica . A 100-bp DNA ladder (product no N32315) was used as a molecular dimension marker to estimate the size of the products.   The antimicrobial susceptibility of the Salmonella enterica isolates was determined according to the Kirby-Bauer Disk Diffusion method on Muller Hinton agar (HIMEDIA, M173, India) . Pure colonies from brain heart infusion agar were removed with a disposable plastic loop, transferred to a tube containing 5 ml of brain heart infusion broth, and emulsified. The emulsified broth culture was incubated at 37°C overnight and compared with 0.5 McFarland turbidity standards. The cotton swabs were immersed in emulsified broth, the contents were smeared evenly on Muller Hinton agar plates, and the operation was performed under a laboratory safety cabinet (level II). The plates were incubated at room temperature for 15 min to allow drying. Antibiotic discs with known concentrations of antimicrobials were carefully placed on plates, and the plates were incubated for 24 h at 37°C. Following incubation, the diameters of the clear zones produced by antimicrobial inhibition of bacterial growth were measured to the nearest millimeter for each disc using a transparent straight-line ruler and then classified as resistant or susceptible according to the Clinical Laboratory Standards Institute . Fourteen antibiotics belonging to different antimicrobial classes were selected based on their clinical and veterinary relevance . The antibiotic agents (Oxoid, UK) used were amoxicillin (30 µg), ampicillin (10 µg), ceftazidime (30 µg), clindamycin (2 µg), ciprofloxacin (5 µg), Ceftriaxone (30 µg), Chloramphenicol (30 µg), doxycycline (30 µg), erythromycin (15 µg), norfloxacin (10 µg), streptomycin (10 µg), vancomycin (30 µg), trimethoprim (5 µg) and tetracycline (30 µg). The multidrug resistance (MDR) (resistance to three and more than three antimicrobial classes) characteristics of the isolates were identified by observing the resistance patterns of the isolates to the antibiotics . A total of 12 PCR confirmed Salmonella enterica positive isolates (3 from environmental, 2 from stool and 7 from milk supply chain) were used for the analysis.   Data entry and management were performed using Microsoft Office Excel 2010, ensuring that all entries were accurately recorded and saved. After thorough data screening to check for errors and completeness, data analysis was performed using STATA version 14 (StataCorp, 4905 Lakeway Drive, College Station, Texas 77845 USA). Descriptive statistics such as frequency and percentage were employed to summarize the data. Chi square(x 2 ) test was used to assess the association between the contamination of Salmonella enterica and independent variables. The multidrug resistance (MDR) characteristics of the isolates were identified by observing the resistance patterns of the isolates to the antibiotics .   Contamination rate of Salmonella enterica Table provides a detailed overview of the contamination rate of Salmonella enterica across various sample types collected from Wolaita Sodo, Southern Ethiopia. The overall occurrence of Salmonella enterica based on the results of bacteriological analysis was 1.86% (12/644; 95% CI: 1.07–3.23). Notably, milk collectors’ milk exhibited the highest contamination rate of Salmonella enterica 6% (3/50, 95% CI: 2.06–16.22) followed by teat surface swabs 1.96% (1/51; 95% CI: 0.35–10.30), bulk tank milk 1.89% (2/106, 95% CI: 0.52–1.62), and equipment swabs 1.82% (1/55; 95% CI: 0.32–9.61). However, the difference in the contamination rate of Salmonella enterica among the different sample types was not statistically significant. When aggregated, the detection of Salmonella enterica was 2.06% (7/339: 95% CI: 1.0–4.20) in the milk supply chain, 1.94% (2/103: 95% CI: 0.53–6.81) in diarrheic outpatients, and 1.49% (3/202: 95% CI: 0.51–4.27) in the milking environments. Serology and PCR confirmation Among the 126 presumptive Salmonella enterica isolates tested using the Latex agglutination test, 45.24% (57/126) of them were tested positive. Out of the 57 serologically positive isolates, 21.05% (12/57) were confirmed as Salmonella enterica using PCR targeting the invA gene. Antimicrobial susceptibility The antimicrobial susceptibility profile of the Salmonella enterica isolates revealed the occurrence of resistance to at least one antimicrobial drug. All the 12 PCR confirmed isolates were resistant to most of the antimicrobial agents: clindamycin (100%), erythromycin (100%), vancomycin (100%), amoxicillin (100%), ceftazidime (100%), ampicillin (75%), streptomycin (67%), doxycycline (67%), ceftriaxone (58%), chloramphenicol (50%), and tetracycline (58%). 92%, 83% and 67% of the isolates were susceptible to norfloxacin, ciprofloxacin and trimethoprim, respectively (Table ). Among the 12 Salmonella enterica isolates tested for antimicrobial classes, all of the isolates were exhibited multidrug resistance for all antimicrobial classes, of which 1 (8.33%) exhibited resistance to 6 antimicrobial classes, 5 (41.67%) exhibited resistances to 7 antimicrobial classes, and 5(41.67%) exhibited resistance to 8 of antimicrobial classes and 1(8.33%) exhibited resistance to 9 of antimicrobial classes (Table ).   Salmonella enterica Table provides a detailed overview of the contamination rate of Salmonella enterica across various sample types collected from Wolaita Sodo, Southern Ethiopia. The overall occurrence of Salmonella enterica based on the results of bacteriological analysis was 1.86% (12/644; 95% CI: 1.07–3.23). Notably, milk collectors’ milk exhibited the highest contamination rate of Salmonella enterica 6% (3/50, 95% CI: 2.06–16.22) followed by teat surface swabs 1.96% (1/51; 95% CI: 0.35–10.30), bulk tank milk 1.89% (2/106, 95% CI: 0.52–1.62), and equipment swabs 1.82% (1/55; 95% CI: 0.32–9.61). However, the difference in the contamination rate of Salmonella enterica among the different sample types was not statistically significant. When aggregated, the detection of Salmonella enterica was 2.06% (7/339: 95% CI: 1.0–4.20) in the milk supply chain, 1.94% (2/103: 95% CI: 0.53–6.81) in diarrheic outpatients, and 1.49% (3/202: 95% CI: 0.51–4.27) in the milking environments.   Among the 126 presumptive Salmonella enterica isolates tested using the Latex agglutination test, 45.24% (57/126) of them were tested positive. Out of the 57 serologically positive isolates, 21.05% (12/57) were confirmed as Salmonella enterica using PCR targeting the invA gene.   The antimicrobial susceptibility profile of the Salmonella enterica isolates revealed the occurrence of resistance to at least one antimicrobial drug. All the 12 PCR confirmed isolates were resistant to most of the antimicrobial agents: clindamycin (100%), erythromycin (100%), vancomycin (100%), amoxicillin (100%), ceftazidime (100%), ampicillin (75%), streptomycin (67%), doxycycline (67%), ceftriaxone (58%), chloramphenicol (50%), and tetracycline (58%). 92%, 83% and 67% of the isolates were susceptible to norfloxacin, ciprofloxacin and trimethoprim, respectively (Table ). Among the 12 Salmonella enterica isolates tested for antimicrobial classes, all of the isolates were exhibited multidrug resistance for all antimicrobial classes, of which 1 (8.33%) exhibited resistance to 6 antimicrobial classes, 5 (41.67%) exhibited resistances to 7 antimicrobial classes, and 5(41.67%) exhibited resistance to 8 of antimicrobial classes and 1(8.33%) exhibited resistance to 9 of antimicrobial classes (Table ).   Understanding the epidemiology of Salmonella enterica along the milk supply chain is important for testing and devising effective interventions to ensure milk safety. Our study investigated the occurrence of Salmonella enterica along the milk supply chain, milking environment and humans and assessed the antimicrobial susceptibility of the isolates in Wolaita Sodo, Southern Ethiopia. Our study demonstrated the widespread occurrence of antimicrobial resistant Salmonella enterica in all sample types collected and tested, raising a serious public health concern for the community. This is particularly a critical public health concern, as milk is mostly consumed in raw and fermented forms in the study area . The 1.5% Salmonella enterica detection in pooled udder milk is lower than the reports from previous studies in different countries that reported ranging from 3.3 to 12.5% , whereas it is significantly lower than that of 21.3% in Ethiopia , 26.3% in Northwest Ethiopia , 64.3% in Modjo, Ethiopia , and 70% in Bahir Dar City, Ethiopia . The detection techniques (culture-based vs. molecular method) and dairy cow feeding and watering habits between dairy farms and countries may increase the variation in the occurrence of Salmonella enterica reported by different researchers . Milk storage containers’ swab in the farms has 1.82% Salmonella enterica which was lower than the findings of 3.3% in Jigjiga, Ethiopia , 14.3% in Sudan , 15.79% in Oromia, Ethiopia  and 25% in Oromia, Ethiopia . The 1.7% of Salmonella enterica in milkers’ hand swabs was lower than the results published elsewhere . No Salmonella enterica was detected in farm floor swabs compared to the 5% in Central Ethiopia , and 11.1% in Central Ethiopia . The differences may be due to the environmental condition, study area, and the laboratory method used for the bacterial identification . The 1.94% of Salmonella enterica in diarrheic outpatients is lower than the findings of 6% in Addis Ababa, Ethiopia , 6.2% in Jimma South-West Ethiopia , 8.72% in Ethiopia , 9% in Middle East , and 10.2% in Tunisia , 11.5% in Harar, Eastern Ethiopia , 13.6% in Addis Ababa, Ethiopia  and 14.28% in Northwest Ethiopia . However, this percentage was significantly lower than the global report by WHO 17.1% , 17.9% in Morocco , 18% in Jimma, south‒West Ethiopia  and 79.6% in South Africa . The differences in the prevalence report could be due to difference in the methodology of detection and the age of the study participants where some of the studies focused on only children and adults and others to all age categories . The observed antimicrobial susceptibility profiles where the majority of the Salmonella enterica isolates were resistant to most commonly used antibiotics are in line with the findings of Noto and Gemeda , who reported the highest resistance to amoxicillin (100%); ampicillin (94.1%) in Haramaya, Eastern Ethiopia , and resistance to amoxicillin, and ampicillin in Southern Ethiopia . Resistance to ampicillin and amoxicillin poses challenges to the veterinary and human health sectors . Similarly, our findings of the susceptibility of Salmonella enterica isolates to norfloxacin (92%), and ciprofloxacin (83%), are comparable with the findings of 82.72% susceptibility to ciprofloxacin in Bangladesh , 81.8% susceptibility to norfloxacin in Addis Ababa, Ethiopia , and 78.3% to norfloxacin in Southern Ethiopia , and 91.7% to ciprofloxacin in Asella, Ethiopia . The 100% multidrug resistance level among the Salmonella enterica strains is similar to the findings of Noto and Gemeda  and Gebeyehu et al.  that reported MDR in all Salmonella enterica . It is nearly comparable to the findings of Mohammed and Dubie  and Mridha et al. , who reported 95.65% and 80.91%, respectively. In contrast, this finding is higher than the 50% report by Beyene et al.  and the 53.9% by Umer Seid et al. . The differences in the findings may be related to differences in the types of antibiotics frequently used in the study area and antibiotic usage. The occurrence and spread of MDR Salmonella enterica uniquely pose a significant challenge to the veterinary and human health sectors . The study has some limitations. First, due to the lack of sufficient funds and laboratory capacity, we were unable to further serotype and sequence the confirmed Salmonella enterica in order to determine the specific serotypes and antimicrobial resistance genes attributed to the observed resistances. Second, due to the nature of the study design, cross-sectional design, and sampling at different time frame from different settings (e.g., dairy farm and clinical settings), a causal relationship between the exposure to contaminated milk and the diarrheic outcome in humans are not possible. Third, the study did not explore the genetic relatedness among the isolates from the different sources and was limited to establishing the directionality of the transmission of the Salmonella enterica between dairy cows and humans and the role of contaminated milk and the environment in mediating the transmission pathways. Lastly, the study limited itself to investigating the potential risk factors that might have contributed to the contamination of milk along the supply chain and the milking environment with Salmonella enterica and the exposure factors for diarrheic patients.   This study revealed a widespread presence of antimicrobial-resistant Salmonella enterica throughout the milk supply chain and milk environment thereby highlighting significant public health concerns, particularly given the prevailing practice of consuming raw and fermented milk in the region. Salmonella enterica was identified in 1.86% of the samples, with higher detection rates being observed in collectors’ bulk milk (6%), teat swabs (1.96%), bulk tank milk (1.89%), milk storage containers swab (1.82%), and hand swabs from milkers (1.7%).The detection rate among diarrheic outpatients was 1.94%. The study also revealed higher levels of antimicrobial resistance, particularly to amoxicillin and ampicillin, despite the fact that the majority of the isolates were susceptible to norfloxacin and ciprofloxacin. The observed 100% multidrug resistance is especially alarming, requiring timely interventions. In order to prevent the transmission and spread of antimicrobial resistant Salmonella enterica , proper hygiene practices, public education, and antimicrobial stewardship programs are highly required. These actions would significantly contribute to the effort toward ensuring the safety of milk and milk products for human consumption. Further exploring the genetic relatedness among the isolates from the different sources, quantifying the public risks associated with milk consumption, identifying and testing cost effective intervention options are needed to reduce morbidity and mortality from Salmonella enterica in the area.   Supplementary Material 1.   