Description of the Study Area
The study was conducted on market egg, farm and cloacae swabs of laying chicken in Jimma town, Oromia regional state, Southwestern Ethiopia.
Study Design
A cross-sectional study was conducted from January, 2018 to November, 2018 to isolate, identify and characterize antimicrobial susceptibility patterns of E.coli species from market egg, farm egg and cloacae swabs of laying chicken in Jimma town.
Sample collecting and processing
A total of 166 samples of egg were collected (83 samples of egg from market and 83 samples of egg from farm to maintain proportionality) were collected and again 83 cloacae swabs were collected from chicken these laid sample of egg at farm.
Of 83 samples of egg from Jimma town poultry farms; 16, 58, 6 and 3 samples of egg and cloacae swab of laying samples of egg were collected randomly after clustering samples for sample sours farm that include Jimma University College of Agriculture and Veterinary Medicine, Jimma University Kito Furdisa Development Enterprise, Emabet and Daniel poultry farm respectively. Eggs were collected in sterile bags as soon as egg laid using sterile glove and transported to the laboratory and cloacae swabs from these laying sample of eggs were collected and swabs were placed in sterile tube contain 10 ml peptone water. Eighty three egg sellers were selected randomly from Jimma town markets randomly and Single egg from each egg seller were collected in sterile bags randomly using sterile glove.
Cloacae swabs were incubated at 37oC for 24 h as soon as samples were collected. Samples of egg were processed according to (Loongyai et at., 2011) in which external shell of eggs were swab with sterile cotton swabs dipped in sterile peptone broth and placed in 10 ml of peptone broth and subsequently incubated for 24 h at 37oC. In order to collect the egg contents, eggs were surface sterilized by immersion in 75% alcohol for 2 min, air dried in a sterile chamber for 10 min, then cracked with a sterile knife. Each egg's content was mixed thoroughly and 1 ml of the mixed egg content was inoculated into 9 ml of peptone broth and incubated at 37C for 24 h.
Isolation and identification of E. coli
A loop from incubated peptone water of egg shells swabbed cloacae swabs and egg content were streaked on MacConkey agar and incubated for 24 hr at 37 °C. Colonies on MacConkey agar having bright pink-red via lactose fermentation were taken and streak on Eosin Methline Bleu (EMB) agar and plates were incubated for 24 hr at 37 °C. Then the plates were examined for the presence of colonies that may resemble E.coli according to the technique recommended by [9]. The organisms showing characteristic colony morphology of E. coli on EMB agar having black golden on the center of colonies were isolated (finger1). Isolated colonies were taken directly from the plate and transferred to nutrient agar for farther identification of E.coli with biochemical test.
Isolated E. coli suspected colonies were biochemically identified according to (Dean et at., 1972) in which Catalase positive, Citrate negative, Indole Positive, Motile, Methyl Red (MR) positive, yellow at both slant and bottom, gas formed and negative for the formation of H2S on Triple Sugar Iron Agar (TSIA) test and Voges Proskauer (VP) E. coli colony were identified(finger 2).
Antimicrobial Susceptibility Patterns of isolated E. Coli
Identified E. coli with biochemical test were taken for antimicrobial patters with sub culturing on nutrient agar medium. Twelve (12) antimicrobial discs of clinically utilizing drug were applied for each isolates with qualitative agar diffusion method (Kirby-Bauer method) by employing Mueller Hinton agar medium [10] (finger 3). Culture of each isolated were compared with 0.5 McFarland turbidity standards. Isolates were swabbed on mueller-hinton agar using sterile swabs.
Antimicrobial impregnated discs were seated on the surface of cultures of Muller-Hinton agar and incubate at 37 °C for 20 h intended for E. coli isolates taste for susceptibility to the antimicrobial using the disk diffusion method according to guidelines set by the clinical laboratory standards institute (CLSI) [11]. For each antimicrobial, mm of inhibition zone was measured by using digital vernier calipers and inhibition zone of each antimicrobial were classified (resistant, intermediate, or susceptible).