Prevalence and Antimicrobial Susceptibility of Salmonella enterica in Milk supply chain, Humans, and milking environment in Woliata Sodo, Ethiopia

doi:10.21203/rs.3.rs-4907956/v1

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Prevalence and Antimicrobial Susceptibility of Salmonella enterica in Milk supply chain, Humans, and milking environment in Woliata Sodo, Ethiopia

https://doi.org/10.21203/rs.3.rs-4907956/v1

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Background: Foodborne disease caused by Salmonella enterica is among the leading causes of death worldwide. Few studies have been conducted on the prevalence of Salmonella enterica contamination along milk supply chain, which is important for reducing public health risks.

The objectives of this study were to estimate the prevalence of Salmonella enterica in milk along supply chain; milking environment (floor, teat surface, and storage containers); milkers’ hands, diarrheic patients and to assess the antimicrobial susceptibility profile of the isolates in Wolaita Sodo, Ethiopia.

Methodology: A cross-sectional study design was used to collect a total of 644 samples from 106 dairy farms, comprising tank bulk milk (n=106), cow’s milk (n=133), milkers’ hand swabs (n=59), teat surfaces (n= 51), 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 isolation and identification of Salmonella enterica were performed following standard bacteriological methods, and antimicrobial susceptibility was tested using the Kirby–Bauer disk diffusion method against 14antimicrobials.

Results: The overall prevalence of Salmonella enterica was 19.6% (126/644; 95% CI: 16.6 –226–22.8). The highest prevalence (28.8%; 95% CI: 17.7 – 42.1) was observed in hand swab samples, while the lowest was observed in bulk milk samples (14.2%; 95% CI: 8.1 – 22.3). The prevalence was 24.3% (49/202; 95% CI: 18.52 – 30.77) in the milking environment, 18% (61/339; 95% % CI: 14.1 - 22.5) in the milk supply chain samples and 15.5% (16/103; 95% CI: 9.2- 23.9) in the diarrheic patient samples. However, the difference in the prevalence of Salmonella among the different sample types was not statistically significant (Chi-squared = 8.12; p = 0.422). Among the Salmonella isolates tested, 89.7%, 88.5%, 77.8%, 77.8%, 66.7% and 63.3% were resistant to amoxicillin, ceftazidime, ampicillin, streptomycin, doxycycline, and tetracycline, respectively. All the isolates were susceptible to norfloxacin, and 81% of them were susceptible to ciprofloxacin. Multidrug resistance (MDR) was detected in 88% (44/50) of the isolates. The MDR was higher at the farm than milk collectors and retailers.

Conclusion and Recommendation: Our study revealed a high prevalence of Salmonella enterica along the milk supply chain, in milking environment, and in humans, suggesting the need for intervention. The isolates were phenotypically resistant to most antimicrobials used in the medical and veterinary practices. Moreover, the antimicrobial resistance profile and MDR indicates the emergence of innovation of drug of choice for the treatment of disease of human and animals. Further studies on the genetic relatedness of the isolates from different sources, quantifying the risk of consumption of milk contaminated withSalmonella entericaand identifying the determining factors are required to design tailored and effective measures to ensure milk safety.

antimicrobial susceptibility

milking environment

milk supply chain

prevalence

Salmonella enterica

Woliata

Foodborne diseases (FBDs) are diseases of an infectious or toxic nature caused by the consumption of food or water [1]. 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 [2]. 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 [3]. 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 [2; 4].

Animal source foods (ASFs), 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 [5; 6]. Dairy products are responsible for 20 disability-adjusted life years (DALYs) per 100,000 people in 2010 [7; 8]. This corresponds to approximately 4% of the global burden of foodborne disease and 12% of the animal source FBD burden. Previous study [9] 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 [8; 10].

The most frequent causes of foodborne illness are diarrheal disease agents [2]. 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 [11]. Most of these FBD agents are known to cause public health problems in mono- and multidrug-resistant organisms (MDRs) [12] [13]. Among all FBDs, diarrhea due to Salmonella infections results in the highest burden, causing 4.07 million DALYs [14]. Dairy products, including raw milk and soft unpasteurized cheese, have been identified as sources of Salmonella enterica [15]. 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 [16].

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 [17]. 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 reasons for the occurrence and associated burden of FBD [18;19 and 20].

Few studies conducted previously in different parts of Ethiopia reported a prevalence of Salmonella enterica ranging from 6.5–20% in dairy products such as milk [21; 22; 23; 24 and 25]. These studies were mainly conducted on milk samples collected at specific points and did not cover the entire milk supply chain. In addition, previous studies did not investigate the potential role of common sources of contamination in the milking environment or assess the antimicrobial susceptibility profile of Salmonella 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 infections associated with dairy farming and dairy products is critically important for reducing public health risks. This study was conducted to estimate the prevalence of Salmonella enterica and determine its antimicrobial susceptibility profile in milk along the supply chain at different critical points and in humans in Woliata Sodo, Ethiopia.

Study area

The study was conducted in Woliata Sodo, southern Ethiopia (Fig. 1), 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 South Nations, Nationalities and People’s Regional State. 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 meters (5,200 and 6,900 feet) above sea level [26]. The average annual rainfall in the area is 1,014 mm [26], and the mean annual temperature is 19.9°C, with monthly temperatures ranging from 17.7°C to 22.1°C [27]. According to the Central Statistical Authority (CSA, 2019), 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 mixed crop zone: livestock and urban dairy systems [28]. The livestock population of the Woliata zone is estimated at 841,729 cattle, 240,315 sheep, 159,362 goats, 228 horses and 1,019,6 poultry [29].

Study populations

The study population included cows, humans, dairy equipment, and dairy farm environments. According to the information taken from the owner/attendants/managers of the farm, lactating cows with no history of recent medication were selected from Sodo Town dairy farms. In this study, involving human subjects, outpatients with a history of diarrhea who visited the Otona Comprehensive Referral Hospital and who had a habit of consuming milk and milk products were selected. Moreover, dairy farm personnel who were directly involved in milking practices were selected purposely and participated in the study. Furthermore, dairy farms that distribute milk directly at least to consumers, regardless of their management system, were considered study participants.

Study design, sample size and sampling techniques

A cross-sectional study design was employed to generate the desired data. The sample size for dairy farms was calculated according to the formula described by Ashram [30], which yielded 100 farms, although 106 different farms were included in this study. Lactating cows within the farm were selected randomly from each dairy farm. From smallholder dairy farms, one cow was selected, while two cows were selected from commercial/cooperative dairy farms. A total of 133 udder milk samples were collected from the 106 dairy farms selected. In addition, at least two samples were collected from milking environment from each selected farm. 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 [31], a 5% margin of error and a 95% confidence level using the formula described by Thrusfield [32], 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. Fifty milks collection centers and 50 milk retailers were also included in this study, and they were selected using simple random sampling techniques. Environmental samples in the present study includes the samples found in the milking environments such as milkers hand swabs; surface teat swabs; floor swabs; equipments used for milking storing milk. Milk supply chain includes the chain where milk reaches the consumers from farm such as milk collectors and milk retailers. Table 1 summarizes the number of samples collected from the different sample sources.

Table 1

the number and type of samples collected and processed for the detection of *Salmonella*
N⁰	Sample type	Number of samples
1	Bulk tank milk	106
2	Pooled udder milk	133
3	Milkers’ hand swab	59
4	Teat surface swabs	51
5	Milk storage containers’ swabs	55
6	Floor surface swabs	37
7	Milk samples from milk collectors	50
8	Milk from milk retailers	50
9	Stool samples from diarrheic patients	103
Total		644

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 [33]. Pooled bulk tank milk samples were taken aseptically 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 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 sampling of human biological traces [34 and35]. The swab tip was first swept once on the surface in slalom-like pattern (a bold graphic cable zigzags down a striped garter stitch back drop). Then, the swab was swept perpendicularly for a second time on the surface to 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), quickly shipped to Wolaita Sodo University, Veterinary Microbiology Laboratory, and stored at + 4°C [35]. 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 grams of freshly voided stool in a clean, dry, and leak-proof disposable stool cup provided by a health professional (nurses) 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 sterilized 10 mL of BPW. The swab samples were shaken for 30 seconds for uniform distribution before transportation.

All the sample-containing bottles were clearly labelled/ coded with data and sample sources using waterproof ink and transported within 12 hrs 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 [36]. Briefly, 25 mL of milk sample was transferred into 225 mL of sterile buffered peptone water (Oxoid, CM 0509) 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) were directly incubated at 37°C for 18 hours. Then, 0.1 mL and 1 mL aliquots were aseptically inoculated into 10 mL of sterile Rappaport Vassiliadis (RV) broth (HIMIDIA) and Muller Kaufmann Tetrathionate (MKTTn) broth (HIMIDIA), respectively. The cultures in RVS broth were incubated at 41.5°C for 24 hrs, and those in the MKTTn broth were incubated at 37°C for 24 hrs. A loopful of culture from the RV and MKTTn broths was streaked onto Xylose Lysine Deoxycholate (XLD) Agar (HIMIDIA) and Hektoen enteric agar (HE) (HIMIDIA) and incubated aerobically at 37°C for 24 hr. 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™), incubated at 37°C for 24 hrs 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 hrs. The results of the biochemical tests were interpreted as described by the International Organization for Standardization guidelines [33; 36].

Serological tests

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 degree of agglutination was examined, and the observed signs of agglutination were considered positive for Salmonella.

Molecular identification

DNA Extraction

Serologically identified Salmonella enterica isolates were re cultured from the stored isolates on selective media and then onto brain heart infusion (BHI) 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 [37 and 38].

PCR was carried out using a Bio-Rad T100 thermocycler in a 25 µl reaction volume. Each reaction contained 2.5 µL of a DNA template, 12 µL of PromegaGoTaq Green Master mix (containing Taq polymerase, dNTPs, MgCl₂, and reaction buffer), 0.81 µL of 10 µM primer Salm3, 0.87 µL of 10 µM primer Salm4 and 8.82 µL of nuclease-free water. 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 [39 and 38]. 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 [40].

Visualization of the amplified PCR products

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 in a glass flask supplemented with 5 µL of red gel (5 mg/mL). DNA was separated at 120 volts for 40 mins. 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 isolates was determined according to the Kirby-Bauer Disk Diffusion method on Muller Hinton agar (HIMEDIA M173, India) [3]. 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 [41]. Fourteen antibiotics belonging to different antimicrobial classes were selected based on their clinical and veterinary relevance [42]. 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) (Table 2). The multidrug resistance (MDR) (resistance to more than three classes of antibiotics among all the tested antibiotics) characteristics of the isolates were identified by observing the resistance patterns of the isolates to the antibiotics [43].

Table 2

Number and sources of *Salmonella* isolates tested for antimicrobial susceptibility.
Type of drug	Milking environment	Stool	Milk samples	Total
Amoxicillin (AMC30)	13	3	12	28
Ampicillin (AM10)	14	2	11	27
Ceftazidime (CAZ30)	10	3	11	24
Clindamycin (CC2)	11	3	12	25
Ciprofloxacin (CIP5)	15	2	10	27
Ceftriaxone (CRO30)	17	2	9	28
Chloramphenicol (C30)	11	3	13	27
Doxycycline (D30)	15	-	14	29
Erythromycin (E15)	15	2	10	27
Norfloxacin (NOR10)	13	4	13	30
Streptomycin (S10)	11	3	12	26
Vancomycin (Va30)	17	2	8	27
Trimethoprim (TM5)	13	2	12	27
Tetracycline (Te30)	13	4	13	30

Data management and analysis

Microsoft Office Excel 2010 was used to enter and save the data, and STATA version 14 (StataCorp, 4905 Lakeway Drive, College Station, Texas 77845 USA) was used to conduct the analysis. Descriptive statistics such as frequency and percentage were utilized to describe the observed data. Chi squared test was used to assess the association between the outcome variable, prevalence of Salmonella and sample type. The multidrug resistance (MDR) (resistance to more than three classes of antibiotics among all the tested antibiotics) characteristics of the isolates were identified by observing the resistance patterns of the isolates to the antibiotics [43].

Prevalence of Salmonella

The overall prevalence of Salmonella based on the results of bacteriological analysis was 19.6% (126/644; 95% CI: 16.6–22.8). The highest prevalence (28.8%; 95% CI: 17.7–42.1) was observed in hand swab samples, while the lowest was observed in bulk milk samples (14.2; 95% CI: 8.1–22.3). However, the difference in the prevalence of Salmonella among the different sample types was not statistically significant (Chi-squared = 8.12; p = 0.42) (Table 3). Taken together, the prevalence of Salmonella was 18% (95% CI: 14.1–22.5) in the milk supply chain, 24.3% (95% CI: 18.5–30.8) in the milking environment and 15.5% (95% CI: 9.2–24) in diarrheic outpatients.

Table 3

Prevalence of *Salmonella* in various sample types in Wolaita Sodo, southern Ethiopia
Sample type	N⁰ tested	N⁰ positive	Prev. (%)	95% CI	X²	p-value
Bulk milk	106	15	14.2	8.1–22.3	8.12	0.42
Equipment	55	11	20.0	10.4–33.0
Teat swab	51	12	23.5	12.8–37.5
Udder milk	133	24	18.1	11.9–25.7
Collector bulk milk	50	10	20.0	10.0–33.7
Floor swab	37	9	24.3	11.8–41.2
Hand swabs	59	17	28.8	17.8–42.1
Retailer bulk milk	50	12	24.0	13.1–38.2
Stool sample	103	16	15.5	9.2–24.0
Total	644	126	19.6	16.6–22.9

Over half (55%, 57/103) of the diarrheic outpatients were female, and 41% (42/103) of them were aged < 5 years. Among the Salmonella enterica-positive patients, 75% (12/16) had a recent history of diarrhea and abdominal discomfort; of these patients, 56% were females younger than 5 years, while 10 were older than 5 years. Among the Salmonella-positive diarrheic outpatients, 12 had a history of consuming either raw fermented or boiled milk. None of the patients had a history of previous treatment for salmonellosis (Table 4).

Table 4

Prevalence of *Salmonella* according to demographic characteristics, milk consumption and clinical symptoms of the diarrheic outpatients (n = 103)
s.n	Demographic characteristics	Type	Number of Participants (%)	Salmonella positive (%) (n = 16)
1	Sex	Female	57(55)	9(56)
		Male	46(45)	7(44)
2	Age	< 5yrs	42(41)	6(37.5)
		5-10yrs	14(13.6)	1(6.25)
		10-15yrs	14(13.6)	2(12.5)
		31-40yrs	24(23.3)	5(31.3)
		> 40yrs	9(8.7)	2(12.5)
3	Mode of consumptions	Raw milk	11(10.7)	2(12.5)
		Raw fermented	27(26.2)	6(37.5)
		Boiled	28(27.2)	5(31.3)
		Raw fermented & boiled	24(23.3)	1(6.25)
		Raw milk & fermented	13(12.6)	2(12.5)
4	Symptoms of the disease	Diarrhea	36(35)	6(37.5)
		Abdominal pain	39(38)	6(37.5)
		Diarrhea and vomiting	19(18)	2(12.5)
		Constipation and nausea	9(8.7)	2(12.5)

Serological and molecular confirmation of Salmonella enterica

Latex agglutination test was performed on 126 presumptive Salmonella enterica isolates identified by bacteriological techniques and 45.24% (57/126) of them gave positive results (Fig. 2). Out of the 57 serologically confirmed isolates 21.05% (12/57) were confirmed to be Salmonella enterica using PCR targeting invA gene (Fig. 3).

The antimicrobial susceptibility profile of the Salmonella isolates revealed the occurrence of resistance to at least one antimicrobial drug. More than half of the isolates were resistant to most of the antimicrobial agents: clindamycin (100%), erythromycin (100%), vancomycin (100%), amoxicillin (89.65%), ceftazidime (88.46%), ampicillin (77.78%), streptomycin (77.78%), doxycycline (66.67%), and tetracycline (63.33%). All the isolates were susceptible to norfloxacin (100%), while 81% of them were susceptible to ciprofloxacin (Table 5).

Table 5

Antimicrobial susceptibility profile of *Salmonella enterica* isolates
SN	Type of drug	Resistant (%)	Susceptible (%)	Total	Antimicrobial classes
1	Amoxicillin (AMC30)	26 (89.66)	3 (10.344)	29	Penicillin
2	Ampicillin (AM10)	21 (77.78)	6 (22.22)	27	Penicillin
3	Ceftazidime (CAZ30)	23 (88.46)	3 (11.54)	26	Cephalosporins
4	Clindamycin (CC2)	26 (100)	-	26	Lincosamides
5	Ciprofloxacin (CIP5)	5 (19.23)	21(80.77)	26	Quinolones
6	Ceftriaxone (CRO30)	16 (59.26)	11(40.74)	27	Cephalosporins
7	Chloramphenicol (C30)	22 (78.57)	6 (21.43)	28	Amphenicols
8	Doxycycline (D30)	18 (66.67)	9 (33.33)	27	Tetracyclines
9	Erythromycin (E15)	29 (100)	-	29	Macrolides
10	Norfloxacin (NOR10)	-	29 (100)	29	Quinolones
11	Streptomycin (S10)	21 (77.78)	6 (22.22)	27	Aminoglycosides
12	Tetracycline (Te30)	19 (63.33)	11 (36.67)	30	Tetracyclines
13	Trimethoprim (TMP5)	16 (59.26)	11(40.74)	27	Pyrimidine
14	Vancomycin (Va30)	27 (100)	-	27	Glycopeptides

Among the 50 Salmonella enterica isolates tested for antimicrobial susceptibility, 24% (12/50) exhibited multidrug resistance (resistance to more than three classes of antibiotics among all tested antibiotics), of which 1 exhibited resistance to all classes of antibiotics tested (2.94%), 1 exhibited resistance to 5 classes of antibiotics tested (2.94%), 3 exhibited resistance to 4 classes of antibiotics (8.82%) and 7 exhibited resistances to 3 classes of antibiotics (20.59%).

Understanding the epidemiology of Salmonella enterica along the milk supply chain important for testing and devising effective interventions to ensure milk safety; our study investigated the prevalence of Salmonella enterica along the milk supply chain, in milking environment and humans and assessed the antimicrobial susceptibility the isolates in Wolaita Sodo, southern Ethiopia. Our study demonstrated the widespread occurrence of antimicrobial resistant Salmonella all sample types collected along the value tested raising a serious public health concern for the community. This particularly a critical public health concerns as milk is mostly consumed in raw and fermented forms in the area.

The 14.2%, prevalence of Salmonella enterica in farm bulk tank milk is comparable to the findings of 10% in Central Ethiopia [44] and in Adama and Modjo, Central Ethiopia [45]. However, the prevalence was higher than the 3% prevalence in Addis Ababa, Ethiopia [46] and lower than the 20% in Jimma, Ethiopia in 2012 [22], 24% [38] in Egypt in 2017, 28.57% [47] in Ethiopia in 2018, and 19% [48] in Ethiopia in 2017. Similarly, the 24% prevalence in the retailers’ bulk milk is higher than the 6% prevalence in Gondar, Ethiopia in 2016 [23] and 6.25% reported in Oromia, Ethiopia [49]. This difference might arise from the degree of milk contamination pooled from different sources, the types of equipment used, and difference in hygienic practices of the milk and handlers.

The 18.1% prevalence in pooled udder milk is higher than the reports from previous studies in different countries that reported a prevalence ranging from 3.3–12.5% [50–60; 24; 25; 46; 47] whereas lower than that of 21.3% in Ethiopia [49]; 26.3% in Northwest Ethiopia [61]; 64.3% in Modjo, Ethiopia [48] and the 70% prevalence reported by Azage and Kibret in Bahir Dar City, Ethiopia [62], and Texas [63]. The time elapsed between milking and the final destination, the temperature maintenance of along the milk supply chain, detection techniques (Culture based vs molecular method) sample type, housing conditions, cattle feed types, and dairy cow feeding and watering habits between dairy farms and countries may increase the variation in the prevalence of Salmonella enterica reported by different researchers.

Milk storage containers’ swab in the farms contains 20% Salmonella enterica which was higher than the findings of 3.3% in Jigjiga, Ethiopia in 2016 [50]; 14.3% in Sudan in 2012 [64]; 15.79% in Oromia, Ethiopia in 2018 [47]; and 25% in Oromia, Ethiopia in 2017 [48]. The current findings of Salmonella enterica from hands of milkers’ swab were 28.8% in contrast, the finding of 0%- 10.7% [44; 47; 48; 65–67] were lower than the present finding. In the current study, Floor swab revealed 24.3% which was comparable to the finding of 23.5% in 2018 [68] however; higher than the finding of 5% in Central Ethiopia in 2022 [44] and 11.1% in Central Ethiopia in 2020 [45]. According to Garedew Conditions for contamination of raw milk at different supply points are due to less hygienic practices in pre-milking udder preparation, sub-optimal hygiene of milk handlers, and unhygienic activities during milking and use of unclean milk storage utensils, contamination of milk during transportation or waiting [69].

The 15.5% of Salmonella enterica in diarrheic outpatients is comparable to the findings of 11.5% [72] in Harar, Eastern Ethiopia; 13.6% [46] in Addis Ababa, Ethiopia and 14.28% [61] in Northwest Ethiopia. However, this percentage was greater than the 6% [73] in Addis Ababa, Ethiopia; 6.2% [74] in Jimma southwest Ethiopia; 8.72% [75]in in Ethiopia; 9% [76]; and 10.2% [77] in Tunisia, whereas the present finding was lower than the occurrence of 17.1% [78]; 17.9% [77] in Morocco, 18% [79] in Jimma, south‒West Ethiopia and 79.6% [80]in South Africa. The differences in the prevalence report could be due to age of the patients some of the studies focused on children, adults, the others to all age categories and the methodology of detection.

The observed antimicrobial resistant Salmonella is in agreement with the findings of [81], who reported the highest resistance to amoxicillin (100%), ampicillin (94.1%) and tetracycline (70.6%) in Haramaya, Eastern Ethiopia and the 100% resistance to ampicillin reported by Gebeyehu, et. al., 2022 in southern Ethiopia [58], where the bacteria were resistant to tetracycline, amoxicillin, and ampicillin. Our findings of the susceptibility of all Salmonella isolates to norfloxacin, and 81% susceptibility to ciprofloxacin, are comparable to the findings of [82], who reported 82.72% ciprofloxacin in Bangladesh, 81.8% norfloxacin in Addis Ababa, Ethiopia [83], and 78.3% in southern Ethiopia [58] and 91.7% ciprofloxacin in Asella, Ethiopia [24].The 88% (44/50) multidrug resistance in Salmonella strains is nearly comparable with the findings of [84], where only one isolate showed mono-resistance; 95.65% of the isolates were resistant to at least one or more antimicrobials [83]; 80.91% of the isolates were resistant in Bangladesh [82]; 100% were resistant in Haramaya, Eastern Ethiopia [81]; and 100% were resistant in southern Ethiopia [58], which is higher than the percentages 50% in Asella, Ethiopia [24] and 53.9% in Central Ethiopia [44]. The differences in the findings may be related to differences in the type of antibiotics frequently used in the study area, differences in awareness about antibiotic usage, etc. The occurrence and spread of antimicrobial resistant Salmonella to commonly antimicrobials poses challenges to the veterinary and human health sectors [81].

In the present study, the antimicrobial resistance profiles of Salmonella enterica were tested for 14 antibiotics commonly used for treating both human and animal diseases. The majority of the isolates were resistant to two or more antibiotics. More than half of the isolates were resistant to most of the tested antimicrobials: amoxicillin 26% (89.7%), ceftazidime 23% (88.4%), ampicillin 21% (77.8%), streptomycin 21% (77.8%), doxycycline 18% (66.7%), and tetracycline 19% (63.3%). This finding was in line with the findings of [81], who reported the highest resistance to amoxicillin (100%), ampicillin (94.1%) and tetracycline (70.6%) and 100% resistance to ampicillin [58], where the bacteria were resistant to tetracycline, amoxicillin, and ampicillin. [24] Additionally, the highest resistance to ampicilin and amoxicillin poses challenges to the veterinary and human health sectors [81]. In the present study, all the isolates were susceptible to norfloxacin, while 81% of them were susceptible to ciprofloxacin, which is supported by the findings of [82], who reported (82.72%) ciprofloxacin, (81.8%) norfloxacin in [83], and (78.3%) in [58] and (91.7%) ciprofloxacin [24].

Among the 50 Salmonella enterica isolates tested for antimicrobial susceptibility, 88% (44/50) showed multidrug resistance (resistance to more than 2 antibiotics). This finding was in line with the findings of [84], where only one isolate showed mono-resistance; 95.65% of the isolates were resistant to at least one or more antimicrobials [83]; 80.91% of the isolates were resistant in [82]; 100% were resistant in [81]; and 100% were resistant in[58], which is higher than the percentages (50%), [24] and (53.9%) [44]. the differences in the findings may be related to differences in the type of antibiotics frequently used in the study area, differences in awareness about antibiotic usage, etc.

In conclusion, the present study revealed a high prevalence of contamination of raw milk by Salmonella enterica suggesting risk of infection in people consuming raw milk. The study also showed mono and multidrug resistant Salmonella to mostly used antimicrobials in the medical and veterinary practices. Multidrug resistance was also observed in the identified isolates indicating the emergence of innovating new drugs of choice. Future study should focus on quantification of the risk of Salmonella enterica in people consuming raw or unfermented milk (e.g. traditional yogurt) potentially harbouring pathogens. Moreover, public education and awareness about the risk associated with raw milk consumption and the importance of hygiene practices at all stages of milk handling from production to consumption are timely needed to ensure milk safety.

Ethics approval and consent to participate:

The study proposal was reviewed and approved by the institutional ethical review committees of Addis Ababa University, the College of Veterinary Medicine (VM/ERC/17/01/12/2020) and Woliata Sodo University, SNNPRs (IRB/41/12/1223). Before the study begins, detailed discussions were made with the participants about the objective of the study and participants were enrolled after obtaining written informed consent and assent.

Consent for publication:

Not Applicable

Clinical trial number:

Not applicable

Availability of data and materials:

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

None

Funding

This work was supported by the Bill & Melinda Gates Foundation and The Foreign, Commonwealth, and Development Office of the UK [Grant Number INV-00845] awarded to Addis Ababa University; Thematic Research of Veterinary Medicine and Agriculture College, Addis Ababa University, Bishoftu; Woliata Sodo University.

Authors’ contributions

SA: Conceptualization; Investigation; Formal analysis; Data Collection; Data Analysis; Writing-original draft, Writing-review & Editing, AZ: Conceptualization; Investigation; Formal analysis; Data Collection; Data Analysis; Writing review & Editing, BM: Investigation; Formal analysis; Data Collection; Data Analysis; Writing-original draft, TS: Conceptualization; Methodology; Supervision; Writing-review & editing, FD: Conceptualization; Methodology; Supervision; Writing-review & editing, The author(s) read and approved the final manuscript.

Acknowledgements

We would like to express our sincere appreciation to the people and agricultural development agents (DEAs), who took the time to answer the survey questionnaire and provide the samples for the study.

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No competing interests reported.

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Prevalence and Antimicrobial Susceptibility of Salmonella enterica in Milk supply chain, Humans, and milking environment in Woliata Sodo, Ethiopia

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BACKGROUND

MATERIALS AND METHODS

Study area

Study populations

Study design, sample size and sampling techniques

Milk sample collection

Collection of swab samples

Stool sample collection

Serological tests

Molecular identification

DNA Extraction

Visualization of the amplified PCR products

Antimicrobial susceptibility test

Data management and analysis

RESULTS

DISCUSSIONS

CONCLUSIONS

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