Prevalence,Antimicrobial Resistance,and Molecular Characterization of Salmonella in Cattle,Beef,and Diarrheic Patients in Bishoftu,Ethiopia

Abstract

Within Ethiopia, there is a lack of information on the genetic relatedness of Salmonella from cattle, beef, and diarrheic patients and its potential transmission from cattle to humans through consumption of contaminated beef. The objective of this study was to assess the prevalence and determine the serotypes, genetic relatedness, and antimicrobial resistance of Salmonella in cattle in two local slaughterhouses, in beef at retail shops, and in diarrheic patients in the only hospital in Bishoftu, Ethiopia. Salmonella was detected in 2.5% (6/240) of cattle samples, in 8.7% (11/127) of beef samples, and in 2.3% (5/216) of the diarrheic patients. Four Salmonella serotypes: Salmonella Typhimurium, Salmonella Eastbourne, Salmonella Saintpaul, and Salmonella Cotham were identified. Salmonella Typhimurium and Salmonella Eastbourne were isolated from cattle and beef, whereas Salmonella Saintpaul and Salmonella Cotham were isolated only from diarrheic patients. Except for serotype Salmonella Saintpaul, all isolates were grouped into five pulsotypes, of which two pulsotypes contained isolates from cattle and beef. Isolates from humans represented unique pulsotypes. Among the 22 Salmonella isolates tested, 95.5% were resistant to at least 1 of the 14 antimicrobials tested. Three Salmonella isolates originating from cattle were multidrug resistant. One human isolate was susceptible to all antimicrobials tested. More specifically, resistance to ampicillin, sulfamethoxazole, tetracycline, tigecycline, and trimethoprim were observed. The most frequently observed resistance was to sulfamethoxazole (90.9%, 20/22) followed by trimethoprim (22.7%, 5/22). The study revealed considerable Salmonella contamination of beef at retail shops, antimicrobial resistance to commonly used antimicrobials, and shared genetically similar Salmonella serotypes between cattle and beef; the link with humans could not be established. Still, the findings of Salmonella in cattle and beef, the propensity of transfer of Salmonella from cattle to beef coupled with the common consumption of raw/undercooked beef are likely to pose public health risk in Ethiopia.

Introduction

Human Salmonella infection is a major public health concern worldwide (Majowicz et al., 2010). It is mainly manifested by gastroenteritis characterized by diarrhea (Lee et al., 2015). Salmonella was estimated to cause 95 million cases, 50,771 deaths, and 3 million disability-adjusted life years globally in 2017 (Stanaway et al., 2019). Salmonella is responsible for 30% of foodborne outbreaks in the United States (Dewey-Mattia et al., 2018). For 2018, a total of 94,203 confirmed salmonellosis cases were reported with a notification rate of 20.1 cases per 100,000 population within Europe (European Food Safety Authority [EFSA], 2019). Mortality rate from Salmonella infection in developing countries is estimated to be 24% higher than in developed countries (Chimalizeni et al., 2010). The majority of the infections are often associated with ingestion of contaminated foods (Hur et al., 2012).

Cattle are a reservoir for Salmonella with a global estimated prevalence of 9%, ranging from 2% in Europe to 16% in North America, whereas for Africa the estimated prevalence was 9% (Gutema et al., 2019). Cattle play a significant role in the epidemiology of zoonotic salmonellosis (Hoelzer et al., 2011). The presence of Salmonella in cattle, contact with infected cattle, and cross-contamination of carcasses during hide removal and evisceration are common sources of Salmonella infection to humans (Cummings et al., 2010). Salmonella in meat and meat products is the highest risk agent/food pairs causing foodborne outbreaks in humans (European Food Safety Authority and European Centre for Disease Prevention and Control [EFSA and ECDC], 2018). Beef contaminated with Salmonella has been indicated as the source of infection in several outbreaks. For example, in the United States, among the 1965 outbreaks of Salmonella where a food vehicle was implicated during 1973–2011, 96 were attributed to beef accounting for 3684 illnesses (Laufer et al., 2015).

In Ethiopia, the Salmonella prevalence was estimated to be 8.7% in children and 5.7% in adults with diarrhea (Tadesse, 2014), 7.1% in cattle (Tadesse and Tessema, 2014), and 10% in beef (Zelalem et al., 2019) based on meta-analysis. A high level of multidrug resistance was also reported in Salmonella isolated from slaughtered cattle (Eguale et al., 2017; Ketema et al., 2018), raising serious concerns of increased transmission risk of resistant strains in the beef supply chain. A recent study conducted in Ethiopia indicated genetic relatedness among Salmonella isolates from humans and animals including cattle that were collected from Addis Ababa and surrounding districts (Eguale et al., 2018). However, this study did not compare the genetic relatedness of Salmonella isolates from beef with that of cattle and diarrheic patients to investigate the potential transmission of Salmonella from cattle to humans through beef consumption.

Establishing any possible transmission and epidemiological association between the occurrence of Salmonella in cattle, beef, and diarrhea in humans is essential to devise control options. Therefore, the objective of this study was to estimate the apparent prevalence, serotype distribution, antimicrobial resistance profile, and genetic relatedness of Salmonella in cattle, beef, and diarrheic patients in Bishoftu, Ethiopia.

Materials and Methods

Study design and sample collection

The study was conducted from June 2017 to May 2018 in Bishoftu town, located in East Shewa Zone, in the central Oromia, Ethiopia. To estimate the prevalence in the three types of samples (rectal content from cattle at slaughterhouses, beef at the retail shops, and stool samples from diarrheic patients), the number of samples to be collected was calculated using the formula of Thrusfield (2005) to estimate prevalence in large populations. Based on Ethiopian data for the types of samples [7.1% in cattle (Tadesse and Tessema, 2014) and 9% in diarrheic patients (Tadesse, 2014)], a maximum expected prevalence of 9% with a confidence interval (CI) of 95% and an accepted error of 4% was used for the calculation. This resulted in the collection of at least 197 units per sample type. We included 240 rectal content and 216 stool samples. For beef samples, it was decided to visit all retail shops selling beef in the town once and to collect one sample from the available beef.

Rectal content samples (at least 25 g) from 240 cattle were collected from the two slaughterhouses (the municipal and the private slaughterhouse, 120 cattle each) in the town. Cattle were brought to slaughterhouses directly from the open market by beef retail shop owners with no information on the origin of the cattle. The two cattle slaughterhouses served the local community and are typically small, processing on average 30 animals per day. Sampling was performed from the available number of cattle at the slaughterhouses using a systematic random sampling technique at the private slaughterhouse: due to small number available during a slaughter day (usually <10) consecutively at the municipal slaughterhouse. Rectal contents were collected on 14 occasions at the municipal slaughterhouse (June–September, 2017) and on 9 occasions at the private slaughterhouse (October–December 2017). The samples were collected from the rectum using rectal gloves after restraining available animals in a crush located in the lairage.

Meat samples were collected from all of the 127 retail shops selling beef in the town (January to April 2018). On average, 10 beef samples were collected once per week. From each retail shop, one pooled sample of beef cuts (at least 25 g, representing ∼200 cm²) from the surface of the exterior of the carcass (fat tissue) and the surface of lean meat available at the time of the visit was collected and placed into a sterile polyethylene bag.

Diarrheic patients of 1 year or older with a history of passing three or more loose or liquid stools visiting Bishoftu hospital were included in the study. Samples were collected from consecutive diarrheic patients identified during each visit at the outpatient wards of the hospital (January–May 2018). One gram of stool sample was transferred into 9 mL buffered peptone water (BPW) from the stool samples submitted to the clinical laboratory of the hospital for routine testing from eligible and consenting patients who agreed to donate a stool sample. Data on age, gender, type of diarrhea, and beef consumption history were recorded during sample collection. All collected samples were transported in an icebox to the laboratory and stored at 4°C until processed.

Ethical clearance was obtained from Addis Ababa University, College of Veterinary Medicine and Agriculture VM/ERC/06/05/09/2017), Ministry of Science and Technology of Ethiopia (3/10/006/2018) and University Hospital Gent, Belgium (2017/0612). During sample collection, all diarrheic patients were informed about the purpose of the study. Samples were collected after obtaining consent; for minors, assent was requested from the children and written consent from their parents/guardians.

Detection and molecular characterization of Salmonella

For the detection of Salmonella, the ISO method 6579-1: 2017 (ISO, 2017) was applied with minor modifications. In brief, 25 g of beef cuts or 25 g of rectal content samples was transferred into a sterile stomacher bag and mixed with 225 mL of BPW (Difco, BD, Sparks, MD). The mixture was homogenized using a stomacher blender for 1 min at 200 rpm and incubated at 37°C for 18 h. The 1 g stool samples that were collected and transported in 10 mL BPW was directly incubated at 37°C for 18 h. After the incubation of the pre-enrichments, 0.1 mL of each culture medium was spotted in three drops onto modified semisolid Rappaport–Vassiliadis medium (Oxoid, Basingstoke, United Kingdom) and incubated at 41.5°C for 24–48 h. After incubation, each plate was examined for the presence of migration zones. A loopful from the edge of a migration zone was streaked onto xylose lysine deoxycholate (XLD; Difco) agar plate and incubated at 37°C for 24 h. Then, XLD agar plates were examined for the presence of suspect Salmonella colonies (colonies with a black center and a lightly transparent zone of reddish color). Suspected colonies (one to two colonies) were transferred onto tryptone soya agar slant (Oxoid). After incubation at 37°C for 24 h, the cultures were subjected to biochemical tests using triple sugar iron agar slants (Difco, BD), lysine decarboxylase test (BBL, BD), and indole test (BBL, BD) for confirmation. Isolates were stored on tryptone soya agar slants (Oxoid) at −21°C for further characterization.

For serotyping, one Salmonella isolate from each positive sample was first clustered using enterobacterial repetitive intergenic consensus (ERIC) PCR as described by Rasschaert et al. (2005). Based on the obtained ERIC profiles, at least one isolate/profile was selected for serotyping according to the Kauffmann–White scheme (Grimont and Weill, 2007) at the Belgian National Reference Laboratory for Salmonella. Furthermore, all isolates were subjected to pulse field gel electrophoresis (PFGE) after digestion with XbaI enzyme (CDC, 2017). Salmonella Braenderup H9812 was used as reference strain. The fingerprints were grouped according to their similarity with Bionumerics 7.6 software (Applied Maths, Biomérieux, Sint-Martens-Latem, Belgium) using the band-based dice coefficient with a 2% position tolerance and unweighted-pair group method using arithmetic averages. Pulsotypes were assigned on the basis of the difference in the presence of at least one band in the XbaI fingerprint and identified by capital letter.

Antimicrobial susceptibility test

All serotyped Salmonella isolates were tested for their antimicrobial susceptibility to 14 antimicrobial agents (with tested concentration range [μg/mL] in brackets): ampicillin (1–64), azithromycin (2–64), cefotaxime (0.25–4), ceftazidime (0.5–8), chloramphenicol (8–128), ciprofloxacin (0.015–8), colistin (1–16), gentamicin (0.5–32), meropenem (0.03–16), nalidixic acid (4–128), sulfamethoxazole (8–1024), tetracycline (2–64), tigecycline (0.25–8), and trimethoprim (0.25–32). Resistance profiling was evaluated based on the minimum inhibitory concentration (MIC) using Sensititre EU surveillance Salmonella/Escherichia coli plates (Thermo Fisher Scientific, Merelbeke, Belgium). The standard reference strain E. coli ATCC 25922 was used as quality control. The tests were performed according to the manufacturer's instructions. European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological breakpoint values were used to categorize the isolates as resistant or susceptible (EUCAST, 2019). For sulfamethoxazole, tigecycline, and colistin, the MIC values of E. coli were used. Isolates exhibiting resistance to more than two different antimicrobial classes were recorded as multidrug resistant (Magiorakos et al., 2012).

Data analysis

Data were recorded in Microsoft Excel spread sheet (Microsoft Corp., Redmond, WA) and imported to STATA version 15.1 (STATA corp., College Station, TX) for statistical analysis. Apparent prevalence of Salmonella was calculated as the percentage of positive samples from the total number of samples tested and presented with 95% CI. The difference in the prevalence of Salmonella among the three sample types and between the slaughterhouses was analyzed using the Pearson's χ² test. A p < 0.05 was considered significant. Antimicrobial resistance and molecular profiles of Salmonella isolates are expressed descriptively using frequency distributions and percentages.

Results

In total, 22 Salmonella positive samples were detected in this study. The apparent prevalence was 2.5% (95% CI: 0.9–5.4), 8.7% (95% CI: 4.4–14.9), and 2.3% (95% CI: 0.8–5.3) in cattle, beef, and diarrheic patients, respectively. There was a significant difference between the prevalence in beef and in cattle and humans (χ² = 10.69, p = 0.005). However, there was no significant difference in the apparent prevalence of Salmonella between cattle at slaughterhouses and diarrheic patients at the hospital (p > 0.05). Salmonella was recovered from 3.4% (95% CI: 0.9–8.5) and 1.6% (95% CI: 0.2–5.7) of cattle rectal content sampled at the municipal and the private slaughterhouses, respectively. The difference was not significant (χ² = 0.79, p = 0.37). Among the diarrheic patients, 57% (n = 216) of them were male. The mean age of the diarrheic patients was 27.5 years (range: 1–82 years). Salmonella was detected from diarrheic patients of 22, 23, 31, 35, and 52 years old; three females and two males.

The Salmonella isolates belonged to four serotypes: Salmonella Typhimurium (eight isolates), Salmonella Eastbourne (nine isolates), Salmonella Saintpaul (four isolates), and Salmonella Cotham (one isolate) (Table 1). Salmonella Typhimurium and Salmonella Eastbourne were isolated from cattle and beef, whereas Salmonella Saintpaul and Salmonella Cotham were isolated from diarrheic patients. The isolates were grouped into five pulsotypes (A–E) (Fig. 1). Both serotypes Salmonella Typhimurium and Salmonella Eastbourne were further divided into two pulsotypes. Two pulsotypes (B and C) contained isolates from cattle and beef. Pulsotypes A and D contained isolates only from beef and cattle, respectively. Pulsotype E contained an isolate from human and the four Salmonella Saintpaul isolates originating from diarrheic patients were nontypable by PFGE.

FIG. 1.

Pulsed-field gel electrophoresis patterns of Salmonella isolates from cattle, beef, and humans in Bishoftu, Ethiopia.

Table 1.

Prevalence and Serotypes of Salmonella in Cattle, Beef, and Diarrheic Patients in Bishoftu, Ethiopia

Source	n	N	Prevalence in % (95% CI)	Serotypes (no.)
Cattle	240	6	2.5 (0.9–5.4)	Salmonella Typhimurium (2), Salmonella Eastbourne (4)
Beef	127	11	8.7 (4.4–14.9)	Salmonella Typhimurium (6), Salmonella Eastbourne (5)
Humans	216	5	2.3 (0.8–5.3)	Salmonella Saintpaul (4), Salmonella Cotham (1)

95% CI, 95% confidence interval; n, number of tested samples; N, number of positive samples.

Supplementary Table S1 shows MIC distributions of the Salmonella isolates. Among the 22 isolates, 21 (95.5%) were resistant to at least 1 of the 14 antimicrobials tested. One Salmonella Saintpaul isolate from a human sample was susceptible to all antimicrobials. Sulfamethoxazole (20/22, 90.9%) and trimethoprim (5/22, 22.7%) resistance was the most frequently observed in the Salmonella isolates regardless of sample source. Cattle isolates were also resistant to up to three other antimicrobials, namely ampicillin (three isolates), tetracycline (two isolates), tigecycline (two isolates). All Salmonella isolates were susceptible to the remaining nine antimicrobials. Three of the six isolates from cattle were multidrug resistant: one Salmonella Typhimurium and two Salmonella Eastbourne isolates were resistant to four and three antimicrobials, respectively (Table 2).

Table 2.

Antimicrobial Resistance Patterns of Salmonella from Cattle, Beef, and Diarrheic Patients in Bishoftu, Ethiopia

Pattern	Resistance pattern	No. of antimicrobials	Origin	No. of resistant serotypes
Pattern	Resistance pattern	No. of antimicrobials	Origin	Salmonella Typhimurium	Salmonella Eastbourne	Salmonella Saintpaul	Salmonella Cotham
1	AMP^SMX^TET^*TGC	4	Cattle	1
2	AMP^TET^TGC	3	Cattle		1
3	AMP^SMX^TMP	3	Cattle		1
4	SMX^*TMP	2	Cattle	1
			Beef	2	1
			Humans			1
5	SMX	1	Cattle		2
			Beef	4	4
			Humans			2	1

AMP, ampicillin; SMX, sulfamethoxazole; TET, tetracycline; TGC, tigecycline; TMP, trimethoprim.

Discussion

The 2.5% apparent prevalence of Salmonella we observed in cattle at slaughterhouses is lower than the pooled prevalence estimate of 7.1% for cattle at slaughterhouses in Ethiopia with a variation from 2.1% to 16.2% (Tadesse and Tessema, 2014). At the global level, a recent study by Gutema et al. (2019) indicated summary estimate of Salmonella prevalence in healthy cattle at 7.1%. Also, in that study a high variation in prevalence (from 0% up to 95%) was observed between studies, especially in those from countries located in North America and Africa.

The observed 8.7% apparent prevalence of Salmonella in retail beef is comparable with the estimated prevalence of 10% with a range from 6% to 12% in beef in Ethiopia (Zelalem et al., 2019). Other recent studies reported prevalence from 3.3% in Thailand (Prasertsee et al., 2019) up to 30% in Ghana (Ekli et al., 2019).

The 2.3% apparent prevalence of Salmonella in diarrheic patients is comparable with the prevalence reported by another study from Ethiopia (Teshome et al., 2019) but much lower than the national estimated summary prevalence in children (8.7%) and adults (5.7%) with diarrhea (Tadesse, 2014). According to recent studies from other countries, prevalence of Salmonella between 0.4% in Guatemala (Arvelo et al., 2019) and 18.8% in Iraq (Kaabi and AL-Yassari, 2019) were reported in diarrheic patients. In this study, Salmonella was isolated from adult patients. The five patients positive for Salmonella reported raw beef consumption behavior and four of them had a history of raw beef consumption within 14 days before the onset of diarrhea.

In this study, Salmonella was detected in only 22 samples of a total of 583 collected samples. From each positive sample, one isolate was further characterized. The Salmonella isolates belonged to four serotypes. Out of the four serotypes identified, Salmonella Eastbourne and Salmonella Typhimurium were isolated from both cattle and beef. Even within each serotype, a common pulsotype was detected in isolates from cattle and beef. Moreover, the prevalence of Salmonella on beef at retail was remarkably higher than in cattle. This demonstrates that a cross-contamination of carcasses during slaughter and transport to and in retail shops may occur. Salmonella Saintpaul and Salmonella Cotham were the two Salmonella serotypes isolated only from diarrheic patients. Based on these limited findings, a link between cattle and beef on one hand and human illness on the other hand could not be established.

Several studies from Ethiopia demonstrated that Salmonella serotypes causing diarrhea in humans were also present in cattle and beef as reviewed by Tadesse and Tessema (2014) and Zelalem et al. (2019), respectively. More specifically, studies in the country reported Salmonella Eastbourne and Salmonella Typhimurium from cattle (Tadesse and Tessema, 2014), Salmonella Typhimurium and Salmonella Saintpaul from beef (Tadesse and Gebremedhin, 2015; Ketema et al., 2018), and Salmonella Typhimurium and Salmonella Saintpaul from diarrheic patients (Eguale et al., 2015). No citable information is available for Salmonella Cotham from cattle, beef, and humans in Ethiopia. Eguale et al. (2018) found that similar genotypes were present within different serotypes isolated from humans and cattle. Moreover, they showed that also same serotypes and genotypes were present in other food-producing animals. Their findings indicate that different animal species can be the source for Salmonella infection in humans in Ethiopia.

Resistance to at least one antimicrobial substance was observed in 95.5% (21/22) of the Salmonella isolates with multidrug resistance in three isolates from cattle. High frequency of resistance to sulfamethoxazole (90.9%) was found, followed by trimethoprim (22.7%) and was present in isolates originating from cattle, beef, and humans and in all four serotypes. Resistance to three other antimicrobials was found in only three isolates for cattle. In comparison with the present data, within the EU the resistance in Salmonella from animals, foods, and humans to sulfamethoxazole was much lower (varying from 30% to 60%), whereas resistance to trimethoprim was rather similar (varying from 8% to 21%) (European Food Safety Authority and European Centre for Disease Prevention and Control [EFSA and ECDC], 2019). Moreover, the latter report listed higher resistance level for ampicillin and tetracycline and indicated that multidrug resistance differs considerably between EU countries. Different studies were performed in Ethiopia testing the resistance to different antimicrobials of Salmonella isolated from humans and cattle and meat thereof. These studies were based on the disk diffusion method and clinical-resistant values, making a relevant comparison with the present result difficult. Nevertheless, data from those studies testing the same antimicrobials and Salmonella serotypes indicate that the resistant level was in most cases higher than in this study (Ketema et al., 2018).

The combination of sulfamethoxazole and trimethoprim, known as cotrimoxazole, is the most commonly prescribed drug (58.7%) for the treatment of acute diarrhea at the Bishoftu hospital, Ethiopia (Tullu et al., 2018). The resistance of the Salmonella isolates to cotrimoxazole was not tested in this study so that no conclusion about the resistance of the isolates to this product can be directly made. However, based on data mentioned in the EFSA and ECDC report (2019), it can be hypothesized that most of the isolates resistant to trimethoprim may also be resistant to cotrimoxazole. This means that the four isolates originating from cattle (one), beef (two), and humans (one) were potentially resistant to cotrimoxazole.

The study has some limitations. One limitation of this study is the lack of linkage among the sample sources due to sampling of cattle at slaughterhouses and beef at retail shops and stool from patients with diarrhea at a hospital at different time periods. Second, rectal contents, beef, and stool samples were collected in only one town within Ethiopia that may not represent the situation in the whole country. Lastly, due to limited number of Salmonella isolates identified in humans (only five isolates), inferences on the presence or absence of genetic relatedness between the isolates from cattle and beef and humans could not be made.

Conclusions

Our study showed apparent prevalence of Salmonella of 2.5% in cattle at slaughterhouses, 8.6% in beef at retail shops, and 2.3% in diarrheic patients and genetic similarity between Salmonella isolates from cattle and beef. There was no correlation between cattle or beef isolates and human isolates, suggesting that other sources may be involved in human infections. It also revealed a high resistance to sulfamethoxazole and to a much lesser extent to trimethoprim, ampicillin, tigecycline, and tetracycline. In this study, Salmonella Typhimurium and Salmonella Eastbourne were isolated from cattle and beef whereas Salmonella Saintpaul and Salmonella Cotham were isolated from diarrheic patients. The presence of Salmonella in cattle, the potential transfer of Salmonella from cattle to beef, and the common habit to consume raw or undercooked beef in Ethiopia can be a risk for humans. Further robust studies are needed to establish the epidemiological link and to identify the sources of infection in humans.

Footnotes

Acknowledgments

We thank the employees of the slaughterhouses and retail shops, and the diarrheic patients for their participation in this study. We also thank Tsedale Teshome, Martine Boonaert, Sandra Vangeenberghe, Hang Zeng, Sjarlotte Willems, and Eline Dumoleijn for technical support.

Disclaimer

Mention of trade names or commercial products by USDA author (G.E.A.) in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by Addis Ababa University and Ghent University under the Special Research Fund (BOF) program for developing countries.

Supplementary Material

Supplementary Table S1

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