Occurrence,Molecular Characteristics,and Antimicrobial Resistance of Escherichia coli O157 in Cattle,Beef,and Humans in Bishoftu Town,Central Ethiopia

Abstract

Escherichia coli O157 is a Shiga toxin–producing E. coli causing disease in humans. Cattle are the primary reservoir of the pathogen. Information regarding the contribution of cattle to diarrheal illnesses in humans through consumption of contaminated beef is scarce in Ethiopia. We collected samples from 240 cattle, 127 beef, and 216 diarrheic patients in Bishoftu town in Ethiopia to assess the occurrence and determine the virulence genes, genetic relatedness, and antimicrobial resistance of E. coli O157. E. coli O157 was detected in 7.1% of the rectal content samples from cattle in slaughterhouses, in 6.3% (n = 127) of the beef samples, and in 2.8% of the diarrheic patients' stool samples. All isolates were positive for eae gene, 24 (77%) of them were positive for stx2 gene (21 stx2c and 3 stx2a), whereas stx1 gene was not detected. Molecular typing grouped the isolates into eight pulsed-field gel electrophoresis pulsotypes with three pulsotypes containing isolates from all three sources, one pulsotype containing one isolate from human origin and one isolate from beef. The remaining four pulsotypes contained isolates unique either to beef or to humans. With the exception of 1 multidrug-resistant isolate from beef, which was resistant to 8 antimicrobial drugs, the remaining 30 isolates were susceptible to the 14 antimicrobials tested. In conclusion, the finding of genetically similar isolates in cattle, beef, and humans may indicate a potential transmission of E. coli O157 from cattle to humans through beef. However, more robust studies are required to confirm this epidemiological link.

Introduction

Diarrheal disease is one of the leading causes of morbidity and mortality globally (Abubakar et al., 2015), accounting for an estimated 1.6 million deaths annually with most occurring in resource-limited countries and in young children (Troeger et al., 2018). Over 90% of the diarrheal deaths occurred in sub-Saharan Africa and South Asian countries (Vos et al., 2016; Troeger et al., 2018). In Ethiopia, diarrhea is among the top five leading causes of mortality. In 2015, the death rate in the country due to diarrhea was estimated at 88.6 per 100,000 people (Misganaw et al., 2017).

Escherichia coli belongs to the normal intestinal microflora of warm-blooded animals and humans (Ewing, 1986). However, some E. coli strains can cause infection such as diarrheal diseases in humans (Nataro and Kaper, 1998). Diarrheagenic E. coli strains are divided into seven pathotypes (Croxen et al., 2013). A subset of pathotype Shiga toxin–producing E. coli (STEC), called enterohemorrhagic E. coli, is associated with bloody diarrhea, hemorrhagic colitis, and life-threatening conditions, including hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura in humans (Croxen et al., 2013). E. coli O157:H7 is the most widely known STEC serotype (Lim et al., 2010).

It was estimated that STEC causes 2,801,000 acute illnesses, 3890 cases of HUS, and 230 deaths in humans annually across the world (Majowicz et al., 2014). Among those, a total of 10,200 acute illnesses of STEC infections occur in Africa with an incidence rate of 1.4 cases per 100,000 persons per year. E. coli O157 was estimated to contribute 10% to these cases in Africa (Lupindu, 2018).

Cattle are the primary reservoir of E. coli O157 (Gyles, 2007). They play an important role in the epidemiology of diarrheal illness in humans, serving as an important source of infection (Griffin and Tauxe, 1991). The most frequent mode of transmission of E. coli O157 to humans is the consumption of contaminated meat and meat products (Croxen et al., 2013). Beef is one of the most important food sources attributed to STEC infection (Pires et al., 2019).

Studies about the occurrence of E. coli O157 in cattle (Abdissa et al., 2017; Atnafie et al., 2017; Haile et al., 2017) and in beef (Assefa, 2019) in Ethiopia are available. With the exception of one study about E. coli in children with diarrhea (Adugna et al., 2015), information on the occurrence of E. coli O157 in diarrheic patients is lacking for Ethiopia. Moreover, there is no information concerning the genetic relatedness and antimicrobial resistance profile of E. coli O157 in cattle and beef and its potential association with human diarrheal illness. Therefore, the objective of this study was to investigate the occurrence and antimicrobial resistance of E. coli O157 in slaughter cattle, beef at retail shops, and diarrheic patients in humans in Bishoftu town, Ethiopia. The genetic relatedness of the isolates from cattle, beef, and human sources was compared to establish any potential transmission of E. coli O157 from cattle-to-beef-to-humans.

Materials and Methods

Settings and sample collection

A cross-sectional study was conducted at 2 slaughterhouses (one municipal and one private), 127 retail shops, and Bishoftu hospital in Bishoftu town located in East Shewa Zone of Oromia regional state of Ethiopia from June 2017 to May 2018.

The two cattle slaughterhouses serve the local community and are typically small, processing on average 30 animals per day. Rectal content samples (at least 25 g) were collected from the available number of cattle at the moment of sampling on 14 occasions at the municipal slaughterhouse (June to September, 2017) and on 9 occasions at the private slaughterhouse (October, 2017 to January, 2018). A total of 240 rectal content samples, 120 from each slaughterhouse, were collected. The samples were collected directly from the rectum using rectal gloves in the lairage before slaughter.

Meat samples were collected from all (n = 127) retail shops in the town. On average 10 meat samples were collected per week. From each retail shop, one pooled sample of beef cuts (at least 25 g) from the exterior of the carcass (fat tissue) and surface of lean beef available at the time of collection was collected into a sterile polyethylene bag.

Diarrheic patients of 1 year or older with a history of passing ≥ three loose or liquid stools visiting Bishoftu hospital were included in the study. Samples were collected from consecutive diarrheic patients identified during each visit at outpatient wards at the hospital. From the stool samples submitted to the clinical laboratory of the hospital for routine testing from qualifying patients, a 1 g stool sample was collected into 9 mL buffered peptone water (BPW) and stored at 4°C until transport. In total, 216 stool samples were collected. All collected samples were transported in an icebox to the laboratory and stored at +4°C until processed.

Detection and characterization of E. coli O157

Twenty five grams of each rectal content and beef cut sample was transferred into a stomacher bag containing 225 mL of modified tryptone soya broth (Oxoid, Basingstoke, United Kingdom) supplemented with 20 mg/L novobiocin (mTSBn; Sigma Aldrich, MO), homogenized using a stomacher blender for 1 min at normal speed (200 rpm) and incubated at 41.5°C for 6 h. The enriched samples were subjected to immunomagnetic separation using Dynabeads anti- E. coli O157 (ThermoFisher Scientific, West Palm Beach, FL) according to the manufacturers' instruction. The final washed bead–bacteria complexes were spread onto cefixime tellurite sorbitol MacConkey agar (Oxoid) containing 0.05 mg/L cefixime and 2.5 mg/L potassium tellurite (CT-SMAC; Oxoid).

For stool samples, after incubating the BPW medium containing the sample at 37°C for 24 h, a loopful was streaked plated onto CT-SMAC. After incubating all CT-SMAC plates at 37°C for 24 h, the agar plates were examined for the presence of suspect colonies. From each selective agar plate, up to three suspect colonies were subjected to indole, Kligler Iron agar, and E. coli O157 latex agglutination (Oxoid) tests.

From each positive sample, one isolate was further tested for the presence of the gene defining the somatic antigen O157 (Wang et al., 2002) and virulence genes coding for Shiga toxins (stx1 and stx2) and intimin (eae) by the multiplex polymerase chain reaction (PCR) protocol described by Botteldoorn et al. (2003). The isolates positive for the gene stx2 were further subtyped using the PCR method described by Scheutz et al. (2012).

Moreover, all the E. coli O157 isolates were genotyped by pulsed-field gel electrophoresis (PFGE) after digestion with XbaI enzyme according to the standardized PulseNet international protocol (CDC, 2017). The obtained fingerprints were grouped according to their similarity with Bionumerics 7.6 software (Applied Maths, Biomérieux, Sint-Martens-Latem, Belgium) using the Pearson coefficient and unweighted-pair group method using arithmetic averages with an optimization of 2%. Pulsotypes were assigned based on their polymorphisms, namely the difference in the presence of at least one band in the fingerprint (Cobbaut et al., 2009).

Antimicrobial susceptibility testing

The antimicrobial resistance of the isolates was evaluated by determining the minimum inhibitory concentration using EUVSEC Sensititre plates (ThermoFisher Scientific). The tests were performed according to the manufacturer's instructions. The following 14 antimicrobial agents were evaluated: ampicillin, azithromycin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, colistin, gentamicin, meropenem, nalidixic acid, sulfamethoxazole, tetracycline, tigecycline, and trimethoprim. The standard reference strain E. coli ATCC 25922 was used as quality control. European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological breakpoint values were used to categorize the isolates as resistant or susceptible (EUCAST, 2019).

Data management and statistical analysis

Data were entered into Microsoft Excel spread sheets (Microsoft Corp., Redmond, WA), imported to and analyzed using STATA version 15.1 (STATA corp., College Station, TX). The occurrence of E. coli O157 was derived as the percentage of culture-positive samples from the total samples tested from each source. The difference in the occurrence of E. coli O157 between the two slaughterhouses was tested using Fischer's exact test. Antimicrobial resistance and molecular profiles of E. coli O157 isolates were expressed using frequency and percentage.

Ethics statement

Ethical clearance was obtained from Addis Ababa University 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 and samples were collected after obtaining written consent, for minors assent was requested from the children and written consent was obtained from their parents or guardians.

Results

E. coli O157 was detected in 17 (7.1%) out of the 240 rectal content samples from cattle. E. coli O157 occurrence was significantly higher (Fischer's exact p < 0.001) in cattle sampled at the municipal slaughterhouse (13.3%, 16/120) than in cattle at the private slaughterhouse (0.8%, 1/120). Eight (6.3%) of the 127 beef cut samples collected at retail shops and 6 (2.8%) of the diarrheic patients were positive for E. coli O157.

Over a half (57.0%, 123/216) of the diarrheic patients were males. The mean age of the diarrheic patients was 27.5 years (range: 1 to 82 years). Of the E. coli O157-positive patients, four had watery diarrhea, whereas the other two had mixed (mucoid and bloody) diarrhea. All positive patients were males; in the group of ≤5 years (n = 22), there was one positive child of 4 years old; in the group of 5–64 years (n = 188), there were four people testing positive aged 26, 29, 35, and 52 years; and in the group of ≥65 years old (n = 6), there was one person of 78 years old. All E. coli O157-positive adult diarrheic patients had a history of raw beef consumption and three of them had consumed raw beef within 14 days before diarrheal onset.

All 31 E. coli O157 isolates were tested for the presence of virulence genes, antimicrobial susceptibility, and genotyping by PFGE. The eae gene was detected in all isolates, and the stx2 gene in 24 (77%) isolates (cattle = 14, beef = 6, and humans = 4), whereas 7 isolates (3 from cattle and 2 each from beef and humans) were negative for the stx2 gene. The stx1 gene was not detected in any of the isolates. Among the 24 stx2-positive isolates, 21 were positive for the subtype stx2c and the other 3 were positive for the subtype stx2a (2 from beef and 1 from human).

Based on the PFGE genotyping results, the 31 E. coli O157 isolates were grouped into eight pulsotypes (A–H) (Fig. 1). Three pulsotypes (D, E, and F) contained isolates from the three sources (cattle, beef, and humans), pulsotype A contained one isolate from two sources (human and beef) and the remaining four pulsotype groups (B, C, G, and H) representing isolate(s) only from one source. One-third (32%) of the isolates shared the predominant pulsotype F and originated from cattle (eight isolates), beef (one isolate), and human (one isolate). The second common (23% of all isolates) pulsotype D was shared among cattle (three isolates), beef (two isolates), and humans (two isolates). All the seven stx2 gene-negative isolates were grouped into the pulsotype D.

FIG. 1.

Pulsed-field gel electrophoresis patterns and virulence genes of Escherichia coli O157 isolates from cattle, beef, and humans in Bishoftu town, Ethiopia.

Of the 31 E. coli O157 isolates, only 1 isolate from beef showed resistance, exhibiting multidrug resistance, being resistant to eight antimicrobials representing five antimicrobial classes: ampicillin, azithromycin, cefotaxime, ceftazidime, colistin, sulfamethoxazole, tetracycline, and tigecycline (Supplementary Table S1). This isolate belonged to pulsotype D and carried only the eae gene.

Discussion

In this study, the occurrence of E. coli O157 in cattle was 7.1%. E. coli O157 occurrence was significantly higher in cattle sampled at the municipal slaughterhouse (13.3%) than in cattle at the private slaughterhouse (0.8%). The season of sampling, origin of cattle, and transportation of cattle from origin to the market and then to the slaughterhouse might have contributed to this difference (Barham et al., 2002; Hussein and Bollinger, 2005). The overall occurrence was comparable with the global prevalence estimate of 5.7% that ranges from 0.1% to 61.8% (Islam et al., 2014). The difference in the occurrence of E. coli O157 in cattle could be attributed to several factors such as seasonal variation, age, type of cattle, diet, and differences in detection methods (Meyer-Broseta et al., 2001).

The E. coli O157 occurrence of 6.3% in beef was comparable with the national prevalence estimate (6%) in Ethiopia (Zelalem et al., 2019). This percentage was higher than in studies in other countries that reported 2.2% in Nigeria (Tafida et al., 2014), 0.3% in European Union (EFSA, 2013), 0.8% in the United States (Hill et al., 2011), and 1.7% in Australia (Kiermeier et al., 2011). The difference in the occurrence of E. coli O157 in beef can be due to differences in the hygienic handling practices during slaughter, transport of beef, and handling and storage of beef at retail shops (Callaway et al., 2009). Beef contaminated with E. coli O157 can potentially lead to diarrheal illness in humans (Heredia and García, 2018), particularly in countries where consumption of raw or undercooked beef is common such as in Ethiopia (Avery, 2004).

E. coli O157 was detected in the stool from 2.8% of patients with diarrhea. Previous studies reported lower prevalence of E. coli O157 from patients with diarrhea such as 0.5% in Spain (Blanco et al., 2004), 0.5% in Tunisia (Al-Gallas et al., 2006), and 1.2% in Bangladesh (Islam et al., 2007). Although the number of patients ≤5 years (n = 22) and ≥65 years (n = 6) was low, one E. coli O157 was isolated from these age categories. These age groups are at higher risk of experiencing illness caused by E. coli O157 and may have more serious complications from the infection (Smith, 1998; Vally et al., 2012).

All 31 isolates were positive for eae gene, whereas stx2 gene was detected in 24 isolates. The predominant occurrence of stx2 genes as the most virulence factor and predictor of infection in humans was reported by previous studies (Chapman et al., 2001; Kawano et al., 2008; Bai et al., 2018). Of the stx2-positive isolates, 13% harbored the stx2a gene, whereas 87% carried the stx2c gene. The stx2a gene has formerly been identified as an independent risk factor for the development of HUS (Brandal et al., 2015; Dallman et al., 2015; Naseer et al., 2017; De Rauw et al., 2018). According to the classification proposed by De Rauw et al. (2018), STEC isolates carrying the stx2a gene and the stx2c gene have a high and a medium risk for HUS development in patients, respectively. This suggests that patients positive for E. coli O157 carrying such stx2 genes may be more at risk to develop HUS. However, no data about HUS cases in Ethiopia are available up to now. Future study on the ability to produce stx2 and the presence of defective stx phages would elucidate the role of E. coli O157 in HUS infection in the country (Rahman et al., 2018).

Among the isolates, seven were negative for stx genes. It was hypothesized that the absence of such virulence genes may be due to the spontaneous loss of stx genes during multiple subculturing of isolates (Vaishnavi et al., 2010; Joris et al., 2011) or the occurrence of inherently stx-negative isolates (Trabulsi et al., 2002; Cobbaut et al., 2009). E. coli O157 strains missing stx genes clustered phylogenetically with E. coli O157 carrying stx gene (Ferdous et al., 2015) and were considered to be atypical enteropathogenic E. coli (Trabulsi et al., 2002; Hu and Torres, 2015).

Within the isolates from cattle, five pulsotypes were detected, of which pulsotypes C and F were dominant in cattle, suggesting that certain pulsotypes may be widely spread in cattle reared in Ethiopia. This is in contrast to other studies showing that no dominant genetic types of E. coli O157 were present in the cattle population (Cobbaut et al., 2009).

The presence of cattle and/or beef and human isolates within the same pulsotype (A, D, E, and F) suggests the occurrence of circulating isolates and a possible epidemiological link between E. coli O157 in cattle/beef and human infections. Indeed, carcass contamination can occur at the abattoir during the slaughter process (Elder et al., 2000) as well as cross-contamination at the retail shops, especially when no hygienic handling practices are applied, leading to human infection when beef is not well cooked (Maruzumi et al., 2005). Given the significance of few cells of E. coli O157 (Caprioli et al., 2005), the carriage of this pathogen by cattle is critically important due to the substandard hygienic practices at the slaughter houses in Ethiopia (Eshetie et al., 2018) and the risk of carcass contamination during slaughter (Callaway et al., 2009).

Our findings indicated that only one isolate from beef showed resistance (and even multidrug resistance) to the tested antimicrobials. This result is in contrast with published data, indicating that E. coli O157 showed in general a variable resistance against limited antimicrobials such as sulfonamides, streptomycin, tetracycline, and ampicillin (Mir and Kudva, 2019).

The study has some limitations. First, the study lacks directionality since we used a cross-sectional study design for cattle in slaughterhouses, potentially originated from different parts of the country, for the beef sampling at retail shops and for the patients with diarrhea at the hospital. Second, fecal, beef, and stool samples were collected in only one city within Ethiopia, which may not represent the situation in the whole country. Lastly, owing to limited number of E. coli O157 isolates recovered from diarrheic patients (only six isolates), the possible risk factors and their causal relationship of cattle isolates with human diarrhea could not be assessed.

Conclusions

E. coli O157 was observed in 7.1%, 6.3%, and 2.8% in rectal content from cattle, beef at retail shops, and human stools from diarrheic patients, respectively. Genetic similarities were observed for a number of E. coli O157 isolates detected in cattle, beef, and humans, suggesting a potential role of cattle in the development of diarrheal illnesses due to E. coli O157 in humans. In Ethiopia, consumption of raw beef in the form of steak (kurt) dipped in plant-based spices or beef tartare (kitfo) made from raw minced beef is very common (Avery, 2004; Seleshe et al., 2014). Consumption of these raw beef products may be an important source for E. coli O157 infections in the country.

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, Sjarlotte Willems, and Eline Dumoleijn for technical support.

Disclaimer

Mention of trade names or commercial products by U.S. Department of Agriculture (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 USDA. 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 (Scholarship and grant number: 01W03916).

Supplementary Material

Supplementary Table S1

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