Isolation and Antimicrobial Sensitivity Testing of Escherichia coli from Fish Meat Retailing Shops of Mekelle City, Ethiopia

The present study was conducted for the period of six months, September, 2016 to February, 2017 so as to isolate and identify Escherichia (E.coli) from fresh fish samples collected from four different retailing shops in Mekelle city and determine the antibiotic resistance pattern of isolated bacteria. Isolation and identification of E.coli and antibiotic sensitivity test were performed following the standard microbiological techniques. The antibiotic resistance patterns of the E.coli isolates were tested against 6 different antibiotics, namely Ampicillin, Amoxicillin, Ciprofloxacin, Norfloxacin, Gentamicin and Streptomycin. Accordingly, out of the total 96 fish samples collected and analyzed, 9(9.4%) were positive for E.coli following the primary and secondary biochemical test results. Out of the four fish meat retailing shops, the highest and statistically significant prevalence (P=0.001) of E.coli was recorded in shop III as 5(20.8%) followed by shop II as 4(16.7%).Furthermore, results of the antimicrobial sensitivity test have shown that all of the isolates were completely significantly (P=0.001) resistant to two antibiotics (Ampicillin and Amoxicillin) and 100% significantly susceptible (P=0.001) to three of the antibiotics (Ciprofloxacin, Norfloxacin, and Gentamicin). Whereas, out of the 9 isolates, 2 isolates (22.2%) were intermediate and 7 isolates (77.8%) were susceptible to Streptomycin. Hence, it can be concluded that fishes from Mekelle shops do contain antimicrobial resistant pathogenic E.coli where the proportion of these isolates significantly (P<0.001) varies from shop to shop. In spite of the lesser practice of their utilization, Ciprofloxacin, Norfloxacin, and Gentamicin were found to be the best antimicrobials to treat E.coli associated infections in the study area. However, it is suggested that post-harvest hygienic management practices are to be practiced along the fish production-marketing chain.


INTRODUCTION
Fish and fishery products are generally regarded as high risk commodities in respect of pathogen contents and other possible contaminants. Biological contaminants such as bacteria constitute the Awot,T., Tehetna,A., Shishay,A., Belayneh,G., Yohannes,H., Tsegay,T and Netsanet,B(MEJS) V11(2): 229-238, 2019 © CNCS, Mekelle University 230 ISSN: 2220-184X major cause of food-borne diseases. These products are susceptible to a wide variety of potentially pathogenic bacteria and are major vehicles for transmission of several bacterial diseases (Maysoon, 2014). The degeneration of quality of fish is accelerated by microorganisms associated with aquatic environment as well as contamination during post-harvest handling. Poor sanitation and hygiene lead to cross contamination and multiplication of microorganisms and hence poor quality fish are presented to consumers (Jhonstone et al., 1994). Food contamination of antimicrobial-resistant bacteria could be a major public health threat, as the antibiotic resistance determinants can be transferred through the pathogenic bacteria causing compromise in the treatment of severe infections. The prevalence of antimicrobial resistance among foodborne pathogens has increased during recent decades (Rowe-Magnus et al., 2002).
Escherichiacoli strains, in particular are candidate vehicles for such problem, not only due to their diversity but also because they exist as common micro-flora of the gastrointestinal tracts of humans and animals (Clements et al., 2012).
In Ethiopia, numerous studies have focused on antimicrobial resistance patterns and genes in E.coli strains isolated from various sources, including food and animals (Birhanu et al., 2008;Yared et al., 2009). However, very few studies focused on isolation of E.coli from fish.
Therefore, the objective of this study was to isolate E.coli from fish samples sold in retailing shops of Mekelle city and to perform antibiotic sensitivity test on the isolated E.coli.

Description of the Study Area
Mekelle is the capital city of Tigray region, Northern Ethiopia and it is one of the six largest cities of the country located at 39 0 29 1 E and 13 0 30 1 N latitude and longitude at a distance of 783 Km north of the capital Addis Ababa. The mean annual rainfall of the city is 628.8mm, with annual minimum and maximum temperatures of 11.8 and 29.9 o C, respectively (TBARD, 2009).
According to the reports of Central Statistical Agency (CSA, 2007), the city has a total population of 215,546 with different feeding habits. Fish is normally consumed in the city with the highest consumption frequency on the fasting days of the Orthodox religion followers.

Sample Size and Sampling Technique
Following a prior assessment for fish meat retailing shops in Mekelle city, it was found that there are several restaurants involved in selling processed cooked fish-meat, however, there are only fewer shops of about 5-6 involved in vending whole-raw and filleted fish-meat in the city. Out of these, only four shops were considered in the current study based up on willingness of the owners to participate in the study. It has been observed that, there was low and intermittent supply of fish to these shops and fish may not be available all the time for sale. Hence, technically, one visit was made every month and four fish samples were randomly collected from each shop for a period of six months from November, 2016 to April, 2017

Sample Collection and Handling
A total of 96 fish samples were collected from four fish-meat retailing shops in Mekelle city.
Approximately, 30g of raw fish-meat samples were collected in sterile polythene zipper pack and transported to Microbiology laboratory of the College of Veterinary Medicine, Mekelle University using an ice box and stored at 4°C until they are further processed. All samples were processed for E.coli isolation within 24 hrs of collection.

Bacterial Isolation and Identification
Isolation and Identification of E.coli was performed following the standard microbiological technique as recommended by Quinn et al. (2011). In brief, fish microbial samples were collected using swabs from different parts of the fish (the skin, gills and other relevant body parts of the fish) and aseptically streaked on nutrient agar plates. Bacterial growths were identified and recorded following 24-48 hours incubation at 37 o C aerobically and a gram staining was performed according to the method described by Quinn et al. (2011). Then, a single isolate colony was picked and streaked on Mac Conkey agar (Oxoid,UK) plates (Carter et al., 1991).
The Pink colored colonies observed after incubation were considered as presumptive for E.coli.
A single isolated colony was picked and streaked on eosin methylene blue Agar medium (EMB) (Oxoid, USA) and incubated for 24hours. Innocula with a characteristic metallic-sheen on EMB agar medium were considered positive for E.coli. Such colonies were taken and added into nutrient broth for further use.

Antimicrobial Susceptibility Test
The antimicrobial susceptibility test was done by agar disc diffusion assay as described by the Clinical and Laboratory Standards Institute (CLSI, 2000). For susceptibility testing, a pure culture of identified E.coli colonies was taken from nutrient agar and transferred into a test tube containing 5ml of a sterile saline solution and mixed gently to make a homogenous suspension.
A cotton swab was used to strike the bacteria across the surface of Muller Hinton agar and wait for about three minutes to dry. Antibiotic discs were then placed on the cultured agar surface using a clean and sterile forceps. The antimicrobial agents used in the current study were

Observational Study
A conjoint observational study was also conducted using a separately developed format.
Observations such as educational and professional backgrounds of fish-meat retailers, status of retaining shops including product handling methods, storage systems of different incoming stocks or fish product batches, keeping conjoint edible products such as chicken, beef and others in same refrigerator, and others has been observed and documented from the individual shops visited during sample collection.

Data Analysis
The outcomes of the study were entered into a Microsoft excel program spread sheet and analyzed using SPSS statistical software (version 16). Descriptive statistics were computed and expressed in terms of frequency and percentages. The Fisher's exact test and Chi square values were used to compute results between percentages and P-values < 0.05 were used as point to assess for any significant difference between variables.

DISCUSSION
Out of a total of 96 samples tested for E.coli, 9(9.4%) of the study fish samples were positive. All the nine isolates were 100%resistant for two antibiotics, Ampicillin and Amoxicillin.
This result showed that these are the most commonly used antibiotics and results in antimicrobial resistance in the study area. While, this study did not investigate the mechanisms underlying the development of antimicrobial resistance in these E.coli isolates, several previous studies have Awot,T., Tehetna,A., Shishay,A., Belayneh,G., Yohannes,H., Tsegay,T and Netsanet,B(MEJS)  established a causal relationship between antimicrobial use in aquaculture and the increase in specific antimicrobial-resistant bacterial strains (Schmidt et al., 2001;Petersen et al., 2002;Ribeiro et al., 2010;Ye et al., 2013). The use of antibiotic in aquaculture was confirmed by different researchers. Agreeably, the World Health Organization (WHO) has reported the use of many antibiotics for therapeutic treatment and prophylactic purposes in fish; consequently, have stated that the increase in antimicrobial resistance due to excess use and misuse of antimicrobials is a public health concern (WHO, 2001 In the present study, the antibiotics Ciprofloxacin, Norfloxacin and Gentamicin were proved to be the best antibiotics for the treatment of E.coli infection since they were highly effective. In agreement with these findings, Immaculate et al. (2012) who have performed antibiotic sensitivity test on E.coli isolated from sea foods such as fin fishes found considerable levels of susceptibility against Ciprofloxacin (91%) and Gentamicin (78%).

CONCLUSION
The present study revealed that E.coli is a contaminant of fish in the study area and its occurrence in fish could represent a risk to the consumers. There is a difference in the occurrence of E.coli between fish shops due to lack of awareness among shop owners and attendants about strict hygienic measures to avoid pathogenic contamination of fish at various stages of production, storage and transportation to safeguard the public health. Furthermore, it revealed that fish from Mekelle shops contains antibiotic resistant E.coli strains. The result warned the need for more strict preventive measures such as regular washing and sterilization of equipment, utensils, and handler hands starting from the point of production to consumption. However, a Awot,T., Tehetna,A., Shishay,A., Belayneh,G., Yohannes,H., Tsegay,T and Netsanet,B(MEJS)  detail scientific study should be conducted to further elaborate the source of contamination along the production-consumption chain of fish.

ACKNOWLEDGEMENTS
The author and his coworkers would like to express their thanks to Mekelle University, Research directorate for funding the research work through recurrent budget. Special thanks also go to the College of Veterinary Medicine for allowing us to use the laboratories and necessary equipments as well. Finally we would like to thank the Fish-meat retailing shops of Mekelle city for their voluntariness and collaborations throughout the research work.

CONFLINCT OF INTEREST
There are no conflicts of interests.