Assessment of extended-spectrum β-lactamase (ESBLs) – producing Escherichia coli from minced meat of cattle and swab samples and hygienic status of meat retailer shops in Jimma town, Southwest Ethiopia

Extended-Spectrum β- Lactamase (ESBLs)-producing Escherichia coli isolates have emerged as a global threat to human health, and have been isolated from human, animal and environment origins. The small but gradually increasing use of third generation cephalosporins in food animal production may be linked to the recent emergence of ESBLs-producing bacteria that associated with cattle, poultry and pigs [1, 2].

Extended-spectrum β-lactamase (ESBLs) is the current most important resistance mechanisms in Enterobacteriaceae. It can reduce the efficacy of modern expanded-spectrum cephalosporin and monobactams drugs, with the exceptions of cephamycins and carbapenems [3]. The emergence and spread of extended-spectrum β- lactamases (ESBLs) among members of Enterobacteriaceae family originating from food-producing animals and pets is a major public health issue worldwide. A conference held in 2010 by the Veterinary Laboratories Agency reported that ESBL-producing Escherichia coli now occurred on 37% of dairy farms sampled a ‘completely unexpected’ finding [4].

The use of antibiotics for growth promotion in livestock and for treatment of diseased animals may lead to the development of considerable resistant bacterial strains, in which it can be transmitted to humans through the food chain [5]. Escherichia coli has been considered a good indicator of the selective pressure exerted by the use of antimicrobials on the intestinal population of bacteria in food animals, and may also be used as a representative of the Enterobacteriaceae to monitor the emergence and changes in proportion of bacteria possessing extended-spectrum β- lactamase (ESBLs) [6‐8].

A fresh red meats contains a high proportion of water and protein which provides a suitable environment for microbial growth [9]. The occurrence of Escherichia coli in foods of animal origin in Ethiopia is high due to many reasons like illegal slaughtering of animals in open fields, unhygienic slaughter practices, and the risk of disease due to this organism is high because of traditional practices of raw meat consumption. However, the precise attribution of animals and their food products as the sources of resistant strains, and the consequences of it on human health were not yet seriously evaluated. Thus, a study is designed to assess the presence of ESBL-producing E. coli from minced meat of cattle and from swab samples which is obtained from different meat retailer shops in Jimma town, Southwest Ethiopia.

Meat passed through different contacts starting from slaughter house until it consumed as food, which increase its chance of antimicrobial contamination. However, little information is available concerning the prevalence of the resistant strains of E. coli in raw meat and environmental samples from meat retailer shops in the study area as well as in the country, Ethiopia. The current study revealed that, a total of 35 biochemically confirmed E. coli isolates were obtained from 21(23.9%) of minced meat samples and 14 (17.5%) of swab samples. Of these, 7 (20%) of them were confirmed as ESBLs- producers. This is the first report presenting data on meat sources of ESBLs- producing E. coli in our setting. Thus, comparison of our results from different countries is may not be feasible due to differences in the study methodology, such as method of enrichment and isolation procedures, differences in sample size and type of sample and how and when it was collected. However, the proportion of ESBL- producing E. coli strains obtained in this study was lower than the study finding in Vietnam [14] and Pakistan [15] and higher than the study finding in China [16]. In addition to the above methodological differences, the observed differences might be also related with the frequent and miss-use practices of third generation cephalosporins in humans and food animal, since the use of these antibiotics greatly linked to the recent emergence of ESBLs-producing bacteria.

The total E. coli isolates obtained in this study is higher than the study finding in Sothern [17] and Eastern [18, 19], Ethiopia and slightly lower than the study finding in different parts of Ethiopia [20‐22]. The difference observed in the prevalence of E. coli could be due to the differences in the sanitary standards of meat retailer shops premises and hygienic practices of meat handlers. The low sanitary standard of meat retailer shops and poor hygienic practices of meat handlers observed in our survey might be contributing to cross-contamination of meat with E. coli isolates.

The occurrence of E. coli in the raw minced meat samples in this study was (23.9%), which was the highest proportion as compared with hand, knife, chopping board and protective clothing swab samples. This is the fact that, fresh red meats contains a high proportion of internal water and protein activities, which provides a suitable environment for microbial growth [9]. Surface contamination on the carcass can be transferred to meat cuts via meat processing equipment as the animal is further processed [9]. When primal cuts of meats are further processed (e.g. minced/ground meat, sausages), microorganisms are homogenized throughout the product. This is why higher proportions minced meat samples were positive for E. coli in this study. A study from Turkey also showed that, almost all of minced meat samples analyzed were contaminated with E. coli [23]. However, there are no regular inspections of meat retailer shops and abattoirs in our setting to protect meat from contaminations. This is might be posing great problems especially because of the wide spread practice of eating raw meat throughout the country.

Intestinal carriage of E. coli, including ESBLs- producing strains in food-producing animals may leads to contamination of retail meat [24]. A study in France indicates a worrisome prevalence of fecal carriage of cephalosporin resistance in cattle, with a higher prevalence of ESBL-producing E. coli at slaughterhouses compared to farms [25]. Improper handling, processing and display of meat at the slaughtering places and at butcher shops can affect the quality of meat which indicated as the presence of cross-contamination [26]. Thus, the presence of resistant strain of E. coli in meat of cattle and swab samples might be due to cross-contaminations either from caecal contents of animals and/or from different contaminated materials and hands of meat handlers.

The summary of our observational checklist survey showed that, most of butcher shops were didn’t fulfill the requirements of WHO and FAO food safety standards [27]. For example, more than half of butcher shops’ floors and walls were not constructed from materials that are easily washable and only 33% of the floors were free of cracks. Although, more than 80% of the butcher shops have ceiling (local name; ), only 30.7% of them were free of dusts and insects. In additions, only 8% butcher shops have insect and dust proof shelf for meat display and the more than 80% of meat retailer shops’ chopping board was made of wooden material with no easily washable surface. Poor hygienic status of butcher shops and unhygienic practice of meat handlers were also reported in other study in Gondar and Somali region of Ethiopia [28, 29].

None of the butchers had taken any form of formal training on meat safety and hygiene. In addition, none of them have had regular disinfection practices of hands, the floor and processing tools and only one fourth of them were use of separate chopping boards (flat wooden tables) and knives for cutting of internal and/or abdominal organs that have contaminated with caecal contents and other parts of the meats. Eighty three percent (83%) of the butchers were handled money with their bare hands during selling of meat. It is well documented facts that unhygienic practice is one of the most important sources of contamination for foods [13, 26, 30]. For instance, simultaneous handling of food and money increases the risk of cross contamination of meat [31]. Thus, the range of activities involved in meat hygiene should be carried out by responsible bodies with the appropriate training on knowledge and hygienic practices of meat handlers.

In the current study, higher resistance rate were observed in ESBLs-producers toward third generation cephalosporin (p –value < 0.05). However, there is no difference in other classes of antibiotics agents tested. In this study 26 (74.3%) of E. coli isolates were showed co-resistance to three or more classes of antibiotics, such as tetracycline, co-trimoxazole and erythromycin plus β- lactam group of antibiotics. The higher rate of multi-drug resistant (MDR) isolates were also reported in other area of Ethiopia [32], Vietnam [14] and Pakistan [15], in which (56.5 to 96.3%) of ESBLs-producing and non- ESBLs-producing E. coli isolates were showed multiple drug resistance for three or more antibiotics. The result of this study, combined with data from previous studies in different countries, suggests that unhygienic practice of food handling and processing is major contributors to the dissemination of not only ESBL- E. coli but also MDR bacteria. Thus, the development of stringent monitoring strategies and the promotion of hygienic meat distribution practices are needed to control the spread of these antibiotic-resistant bacteria.

In the present study, lower resistance was observed against gentamycin, neomycin and ciprofloxacin and no resistance was seen with amikacin. In contrast, all E. coli isolates of meat and swab samples were resistant to ampicillin, penicillin and erythromycin and more than 50% of the isolates were resistant to tetracycline and co-trimoxazole. This finding is comparable with the previous reports in Eastern part of Ethiopia, in which the prevalence of resistance among E. coli isolates of meat to penicillin, ampicillin, erythromycin and tetracycline were ranging from (33.3 to 100%) [19, 22]. This situation has dragged our condition towards increased load of antibiotics, poor clinical outcome and limited therapeutic options.