Isolation and Antimicrobial Susceptibility Profile of Escherichia coli O157 :H7 from Raw Milk of Dairy Cattle in Holeta District, Central Ethiopia

A cross-sectional study was conducted in small, medium, and large-scale dairy farms of Holeta district to isolate, identify, and antimicrobial susceptibility profile of Escherichia coli O157 :H7 in raw milk of dairy cattle. A total of 210 lactating cows were selected for raw milk samples, and 19% (40/210) were found to be positive for E. coli whereas 5.2% (11/210) were confirmed as E. coli O157 :H7 positive using the Escherichia coliO157 latex test. Accordingly, all E. coli was highly susceptible to Ciprofloxacin (100%), Gentamycin (100%), Oxytetracycline (100%), and Tetracycline (63.63%). Furthermore, the resistance of 72.73%, 54.54%, 54.54%, and 45.45% was developed to Cefoxitin, Sulphamethoxazole, Cloxacillin, and Streptomycin, respectively. Factors such as parity, age, body condition, herd size, milk yield, udder hygiene, and udder lesion showed a statistically significant (p< 0.05) association with the occurrence of E. coli infection in dairy cattle. In conclusion, in this study, a higher prevalence of Escherichia coliO157 :H7 and its drug susceptibility profile is an alarm for the health of the public, and awareness creation to the farm owners and the community is recommended.


Introduction
Milk and dairy products are consumed by billions of people around the globe daily and have valuable nutritional factors such as proteins, lipids, minerals, and vitamins [1,2]. Accordingly, it is important to ensure the microbial quality of milk and dairy products [3]. Foodborne diseases are an important challenge in public health; in particular, developing countries are largely affected because of the prevailing poor food handling and sanitation practices, inadequate food safety laws, weak regulatory systems, and lack of financial resources [4,5]. Foodborne diseases that are caused by bacteria include pathogenic Escherichia coli, Salmonella, Campylobacter, Listeria, Yersinia, Shigella, and Enterobacter, whereas Escherichia coli and Salmonella species were recorded as a major cause of foodborne diseases and food poisoning [6,7].
Escherichia coli O157 : H7 is the most important bacterial pathogens that cause life-threatening infections such as hemorrhagic colitis (HC), abdominal pain, bloody diarrhea, hemolytic uremic syndrome, and kidney failure particularly in humans worldwide [8,9]. Milk and other dairy products are mostly contaminated with E. coli O157 : H7 during direct exposure to feces due to poor handling systems and causes intestinal or extraintestinal disease [10,11]. e high prevalence of E. coli O157 : H7 in dairy products may be due to improper milking hygiene, poor house hygiene, lack of postmilking teat dipping and practicing of milk by contact labors use of lubricant, and absence of order in milking cows of different ages. Moreover, its occurrence was high in dairy farms without being noticeable in farm treatment [12].
Shiga toxin-producing E. coli (STEC), also called verotoxin producing E. coli, are those strains of E. coli that produce at least one member of a class of potent cytotoxins called Shiga toxin [13,14]. Numerous sporadic infections and outbreaks caused by Shiga toxin-producing Escherichia coli O157 : H7 (STECO157) have been reported in the United States and elsewhere worldwide. e majority of STEC O157 infections are foodborne; many are associated with bovine sources. STEC O157 was first linked to outbreaks of severe bloody diarrhea in 1982 and is often referred to as a "recently emerged" human pathogen [15]. Enterohemorrhagic E. coli (EHEC) strains are a subtype of the Vero (Shiga) toxin (Vtx or Stx)-producing E. coli (VTEC or STEC) [3,16].
In Ethiopia, the consumption of raw (unpasteurized) milk is very traditional, and contaminated milk and milk products are the most common transmission pathway of E. coli O157 : H7 from animals to humans. Even though the disease caused by E. coli is very important in the country and of great public health concern, E. coli O157 : H7 has received very little consideration in many of the previous public health studies. Most of the previous studies circulated in limited areas and fail to represent the incidence of E. coli O157 : H7 under different management and ecological situations. erefore, this study was conducted to isolate and identify the antimicrobial susceptibility pattern of E. coli O157 : H7 from raw milk of dairy cows in and around Holeta dairy farms.

Study Area.
e study was conducted in selected dairy farms in Holeta town from December 2018 to June 2019. Holeta is located in central Ethiopia, in Oromia National Regional State, West Showa zone, at a distance of 29 km from Addis Ababa. Its astronomical location is latitude/ longitude: 9°00′ N and 38°30′ E longitude and has an altitude of 2400 m above sea level. It has an experience of a bimodal rainfall pattern in which the main rainy season occurs between June and September and the Short rainy season from March to May. e average annual rainfall is about 1144 mm [17].

Study Animals.
e study animals were lactating cows in selected farms and villages of Holeta town. e sampling units were individual lactating cows within a farm under study. Lactating local (indigenous) zebu, Holstein Friesian, and Holstein Friesian cross with local zebu breed in small, medium, and large-scale dairy farms of Holeta town and the surroundings were the study population based on their management systems and owner's willingness.

Study Design and Sample Size.
A cross-sectional study was conducted in the Holeta district. e list of dairy owners was obtained from the concerned body, and the farms were then classified as small, medium, and large based on the number of cows and farms and households selected by simple random sampling from the list while keeping the proportions close to each other as much as possible. All lactating dairy cows in the selected farms were included in the study and a total of 210 cows were sampled from sixteen dairy farms selected by a simple random sampling method.

Study Methodology
2.4.1. Method of Data Collection. A questionnaire was developed, and all information related to the study objectives was recorded. Data collected include address and are pertinent to cow-level factors, including dairy cows age, parity, breed, presence of a lesion on the skin of udder or teat, and milking practice, where the owners of cows wash their hands and udder before and after milking, wash hand and udder before milking, dry udder before milking, and wash hand only before milking; the type of milking and type of floor were obtained by observation and by interviewing the different farm attendants and owners. e age of the animals was determined from birth records and dentition characteristics and categorized as a young adult (3 to 6 years), adults (>6 to 9 years), and old (>10). Parity was categorized as few (1-3 calves), moderate (3-6 calves), and many (6 and above calves).

Milk Sample Collection and Culturing.
A total of 210 fresh milk samples were collected in sterilized containers (bottles) and labeled based on temporary ID given to a cow, kept in a thermos flask icebox, and transported to Addis Ababa University College of the veterinary medicine microbiology laboratory to culture the milk samples for isolation and identification of E. coli O157 : H7. e examination of milk samples was conducted within 5 hours after collection. All milk samples were processed bacteriologically, and different biochemical tests were performed according to the procedures employed by Quinn et al. [18].

Isolation and Identification of Bacteria.
e isolation and identification of E. coli O157 : H7 was performed using techniques recommended by Quinn et al. [18].
(1) Culturing. A sterile loop was dipped into a thawed milk sample and streaked onto MacConkey agar plates as differential media for the identification of E. coli and incubated at 37°C for 24 hours. e presumed well-selected typical and atypical colonies were again subcultured on selective medium Eosin Methylene Blue (EMB) Agar and incubated at 37°C for 24 hrs. Morphologically typical colonies were producing metallic sheen [19] and under the same conditions to get pure colonies of E. coli. After the next 24 hrs of incubation, a well-isolated colony was selected and subcultured further onto nutrient agar (NA) to be used for biochemical confirmation.
(2) Biochemical Confirmation of E. coli. For primary and secondary biochemical tests, pure cultures of a single colony from MacConkey agars were transferred onto a nutrient agar plate. Tests such as Oxidase, Catalase, Indole, Methyl red, Voges-Proskauer (VP), and Citrate (IMViC) tests were done to confirm the presence of E. coli in the test samples. Colonies that are positive for tryptophan utilization (indole test) (red ring), positive for Methyl red, negative for citrate utilization (green slant), and negative for Voges-Proskauer (VP) test were considered to be E. coli positive. [18]. Moreover, the Gram staining of the bacterial colony was done on a sterile glass slide as described by Cheesbrough [20]. Isolates of presumptive E. coli for all biochemical tests were cultured on sorbitol MacConkey agar for further test on latex agglutination test.

(3) Screening Test by E. coli O157 Latex Agglutination Test.
Latex agglutination test was employed using latex kit for the screening of E. coli O157 : H7. Sorbitol-negative (clear) colonies exhibiting colony morphology typical for Escherichia coli O157 : H7 per plate were picked and spread plated on Cefixime tellurite sorbitol MacConkey (CT-SMAC).
en, after 24 hours of incubation, a fresh single colony of nonsorbitol fermenter from sorbitol MacConkey agar was picked and subjected to latex agglutination using an E. coli O157 latex kit. Isolates of presumptive E. coli O157 : H7 for all latex agglutination tests were cultured on nutrient agar (NA) for antimicrobial susceptibility testing.

Antimicrobial Susceptibility Testing.
e antibiotic susceptibility tests of the E. coli O157 : H7 isolates were performed according to the National Committee for Clinical Laboratory Standards (NCCLS) [21] method using Kirby-Bauer disk diffusion test on Muller-Hinton agar. e agar disk diffusion method has been used to test common fastgrowing bacterial pathogens and is recognized to work well with E. coli O157 : H7. Reliable results can be obtained with disk diffusion tests that use a standardized methodology and zone diameter measurement correlated with minimum inhibitory concentration (MIC), and the inhibition zone was measured and interpreted as susceptible, intermediate, and resistant based on the standard inhibition zone given by clinical and laboratory standard institute (CLSI) [22].
Pure colonies on nutrient agar were taken with a wire loop and transferred to a tube containing 5 ml of Saline water and emulsified. e broth culture was incubated at 37°C for 4 hours until it achieved the 0.5 McFarland turbidity standards. A sterile cotton swab was dipped into the suspension, and the bacteria were swabbed uniformly over the surface of the Muller-Hinton agar plate within a sterile safety cabinet. e plates were held at room temperature for 15 minutes to allow drying. Antibiotic discs with a known concentration of antimicrobials were placed, and the plates were incubated at 37°C for 18-24 hours. Eleven different families of antibiotics (Oxoid, UK) were used based on the trend of using these antibiotics in the different farms of the study area (Table 1).

Data Management and Statistical Analysis.
e data collected were entered into the Microsoft Office Excel 2016 spreadsheet and analyzed using STATA version 13. Descriptive statistics were used to summarize the generated data, and the prevalence of E. coli O157 : H7 related to specific risk factors (herd size, type of farm, udder lesion, milk yield, type of floor, parity, milking type, body condition score, age, farm hygiene, and udder hygiene) was determined as the proportion of affected cows out of the total sample. e association of these specific variables on the prevalence of E. coli O157 : H7 was calculated using a chisquare (X 2 ) test. Besides, antibiotic efficacy was determined by comparing the zone of inhibition of each drug with the standard. A statistically significant association between variables is considered to exist if the p value is <0.05.

Isolation and
Identification of E. coli O157 : H7. E. coli isolates grown on MacConkey agar, Eosin Methylene Blue (EMB) Agar, and Sorbitol MacConkey Agar were identified based on different characteristics on biochemical tests [18]. Catalase, Simmon's citrate agar, sugar fermentation on Triple Sugar Iron Agar, Indole Production, Voges-Proskauer, and Methyl red tests were performed. e isolates of the above media were tested by Triple Sugar Iron Agar (TSI) slant culture by stab method, and after 48 hours of incubation at 37°C, yellow slant with yellow butt, presence of gas bubbles, and absence of black precipitate in the butt were observed which is indicative of E. coli. Performing Simon's citrate test, no color change from green to blue was observed, performing Methyl red test, red color indicative of acid production was observed, performing indole test, red ring at the top of culture broth was observed, and performing Voges-Proskauer test, no color change to pinkish was observed.
e serological test was also employed by using the latex agglutination test for screening of E. coli O157 : H7, and then, from 40 E. coli positive samples, eleven isolates were agglutinated in both tests and taken as E. coli O157 : H7. Finally, out of 210 raw milk samples of dairy cows examined for the presence of E. coli O157 : H7 by detailed bacteriological and serological examinations, the results showed that the overall prevalence of E. coli was 19%, out of which 5.2% was E. coli O157 : H7.

Risk Factors Associated with the Occurrence E. coli O157 :
H7. In this study, eleven factors were considered as potential risk factors for the occurrence of E. coli O157 : H7 in the study area. ese were herd size, type of farm, udder lesion, milk yield, type of floor, parity, milking type, body condition score, age, farm hygiene, and udder hygiene.
In the present study, age was taken into consideration, and the prevalence of E. coli O157 : H7 was measured for different age groups of lactating cows. e highest prevalence (82.5%) was found in lactating cows of ages 3-6 years, followed by cows of ages 6-9 years (12.5%), and the lowest prevalence was recorded in cows of ages less than or equal to 9 years old. Parity is also considered as the main factor in the study, and a higher prevalence (87.5%) was recorded in cows that gave birth to 1-3 calves, followed by cows which gave birth to 4-6 calves (12.5%), and the lowest prevalence was recorded in cows which gave birth to greater than 6 calves ( Table 2).
In the current study, milking practices such as washing and drying of udder also have a significant role in the highest prevalence of E. coli O157 : H7. ere was more likelihood occurrence (92.5%) in poor udder hygiene cows as compared to those who wash their cows regularly (7.5%). E. coli O157 : H7 prevalence was also significantly higher in cows with poor body condition (55%) than cows with moderate (40%) and good body condition (5%). e effect of udder lesion on the current prevalence of E. coli O157 : H7 was also studied, and the result revealed that 50% of cows having infected udder were found to be infected with this bacteria. Herd size also played a vital role in the occurrence of E. coli O157 : H7 in the study area, and the prevalence was also significantly higher in farms having more than 42 herd size (50%) than farms with 10-42 herd sizes (37.5%) and less than 10 herd sizes (12.5%) ( Table 2).
As indicated in Table 2, herd size, age, type of farm, udder or teat lesion, type of floor, parity, milking type, body condition score, farm hygiene, and udder hygiene were found statistically significant with a p value less than 0.05.

Discussion
e present study was conducted to assess the prevalence of E. coli O157 : H7 from raw milk of dairy cattle and to determine the antimicrobial susceptibility profile of E. coli O157 : H7 in and around Holeta districts of West Shewa zone, Oromia Regional State of Ethiopia. e potential risk factors associated with the occurrence of E. coli O157 : H7 were explored, and the contribution of E. coli O157 : H7 as important milk-borne pathogens was illustrated using latex agglutination test using anti-O157 and H7 serum.
According to the report of Ranjbar et al. [30] in Iran, the prevalence of E. coli strains in raw milk and traditional dairy product samples was 30.16%. e report of Ombarak et al. [31] in Egypt also showed that the prevalence of E. coli strains in various types of dairy products had a range of 21 to 77% and both were higher than our findings. e overall prevalence of E. coli strains in raw milk samples of studies conducted on Switzerland [32], Iran [33], Italy [34], Egypt [35], Turkey [36], China [37], and Spain [38] had ranged between 1% and 27% which were comparable with our reported prevalence rate.
ere were some probable explanations for the high prevalence of E. coli and E. coli O157 : H7 from raw milk of dairy cattle in our study. At first, Ethiopian farmers especially smallholders used their hands and also traditional equipment for milking procedures which increase the risk of transmission of bacteria into the milk and dairy products [29,39,40]. Second, the lack of maintenance of raw milk samples at temperatures below 4°C facilitates the survival and proliferation of bacteria [24]. irdly, the occurrence of the disease, unhygienic manner of animals, and house floor might have contributed to the environmental contamination of milk with fecal and infected animal wastes [41]. e transmission of pathogenic agents from the infected staffs of the milking halls and also farms is also another potential risk factor [30].
Among the potential risk factors considered, age, parity, body condition, udder hygiene, herd size, and udder lesion found a statistically significant effect p − < 0.05). e prevalence of various livestock infections generally increases with increasing the herd size. Besides, this indicates the lack of knowledge of the community regarding animal husbandry and the large size can affect the health of animals. Age was also found to be associated with the occurrence of E. coli infection, which is higher in animals of old age (≥9) than in younger ones. is could probably associate with the ability of the immune system of an animal to defend against infection-causing agents. e finding of this study was also assessed for the number of parity as a predisposition to E. coli infections [42].
In this study, the prevalence of E. coli O157 : H7 was higher in cows with dirty udder than that of cows with cleaned udder. Dirty udder contributes to a favorable condition for the multiplication of both contagious and environmental pathogens. Udder hygiene was also found to be statistically significant with the incidence of E. coli infection. e other important factor for the occurrence of E. coli infection in this study was the body condition of the animals. Poor body condition was the major risk factor for increased E. coli O157 : H7 prevalence, and this factor remained statistically significant (p − <0.05). is could be associated with the reduced defense status of the animals. Several factors may contribute to poor body conditions including malnutrition or parasitic infection and old age compounded with reproduction stresses [2,12,29].
Antibiotic-resistant bacteria pose a growing problem of concern worldwide. e development of antibiotic resistance among bacteria such as E. coli poses an important public health concern. e effectiveness of treatments and the ability to control infectious diseases in both animals and humans may be severely hampered since the bacteria can be easily circulated in the environment. A relatively high number of strains are resistant to the antimicrobial commonly used in the therapeutic protocol of many humans and animal infections [43]. Food contamination with antibioticresistant bacteria can also be a major threat to public health, as the antibiotic resistance determinants can be transferred International Journal of Microbiology to other pathogenic bacteria, potentially compromising the treatment of severe bacterial infections. e prevalence of antimicrobial resistance among foodborne pathogens has increased during recent decades [44].
In the present study, all of the eleven isolates were highly susceptible to Oxytetracycline, Ciprofloxacin, and Gentamycin followed by relatively lower susceptibility to Tetracycline, Vancomycin, and Trimethoprim. On the other side, the current study revealed that all isolates were highly resistant to Cefoxitin, Sulphamethoxazole, Cloxacillin, and Streptomycin with a proportion of 72.73%, 54.54%, 54.54%, and 45.45%, respectively. e result of this study was almost comparable with the work of Bedasa et al. [26]. Momtaz et al. [3] reported that resistance against Tetracycline (86.88%) and Streptomycin (54.91%) was the most commonly reported antibiotic resistance-based finding of STEC strains of diarrheic patients which were comparable with the current finding.
According to Ranjbar et al. [30] in Iran, E. coli strains (6.72%) were prevalent in hospital food samples whereas Shiga toxin-producing E. coli (STEC) strains were resistant against Tetracycline. In contrast to our finding, Gentamycin and Ciprofloxacin were found resistant to STEC. On the other hand, Stewardson et al. [45] reported that Extended-Spectrum-Beta-lactamase-producing-Enterobacteriaceaeproducing E. coli (ESBL-PE) was the most commonly detected (44.77%) in food samples, and ESBL-PE strains were susceptible to Gentamicin and Ciprofloxacin. Similarly, the finding of Mahanti et al. [46] in India [28] revealed that a high prevalence of resistance against Gentamicin was seen by STEC, Kang et al. in Korea [30] also showed that a high prevalence of resistance against Tetracycline and Streptomycin was detected by STEC, and Castillo et al. in Mexico [47] also showed a high prevalence of resistance against Trimethoprim and sulfamethoxazole by STEC. e variation in resistance for a single drug may be due to the expression of resistant gene coded by the pathogen which is associated with emerging and reemerging aspects of the isolates with regard to different agroecology [48]. is might be due to the inappropriate use of antibiotics for the treatment of diseases or irrational use of antimicrobials for therapeutic and prophylactic treatment [49].

Conclusion
In the present study, E. coli O157 : H7 was the major contaminant of raw milk of dairy cattle in and around Holeta. In the study area, factors such as age, parity, body condition, herd size, udder hygiene, and lesion contributed to microbial contamination of dairy milk. Detection of E. coli in milk is of public health concern due to its zoonotic potential. All tested isolates were susceptible to Ciprofloxacin, Gentamycin, and Oxytetracycline whereas the isolates were found to be resistant to Cefoxitin, Cloxacillin, Tetracycline, Vancomycin, Chloramphenicol, Streptomycin, Sulphamethoxazole, and Trimethoprim. In conclusion, awareness creation to the dairy farmers and all stakeholders at different levels regarding milk handling practices should be given to reduce the milk rejection rate because of spoiled milk and milk-borne pathogens resulting from contamination of milk. It is recommended that veterinary/extension services be provided to livestock farmers on proper animal husbandry and control of zoonotic animal diseases. Most human diseases are caused by pathogens from animal and/or animal products like milk and milk products. However, the contaminated one acts as a source of E. coli O157 : H7 which needs preventive actions at any point in the food production chain. Besides, different epidemiological factors that interplay in E. coli occurrence should be studied routinely. Furthermore, there should be regular antibiotic sensitivity testing to E. coli O157 : H7 to select effective antibiotics and also help to reduce the problem of drug resistance development toward commonly used antibiotics.
Data Availability e data will be provided upon request from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.