E. coli are commensals that inhabit the Gastro Intestinal Tract of (GIT) of healthy animals and human. This bacterium belongs to the family Enterobacteriaceae [1].The organism is gram negative; rod shaped with 2.0 µm long and 0.25-1.0 µm diameter can survive on a variety of substrates. It can utilize mixed acid fermentation in anaerobic condition, producing lactate, succinate, ethanol, acetate and carbon dioxide [2]. The bacterium is classified into different serotypes based on presence of pathogenic flagella antigens. Chin(2000) identified six pathotypes of E. coli namely: Diarrhoeagenic E. coli (associated with diarrhoea), Shigatoxin producing E. coli (STEC) which shared homology to the cytotoxin produced by Shigella dysenteriae, verocytotoxin producing E. coli (VTEC), Enterohemorrhagic E. coli (EHEC), associated with food poisoning, Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (ETEC) and diffusely adherent E. coli (DAEC). The 16S rRNA based phylogenetic analysis has shown close genetic relatedness of E. coliwith other members of the Enterobacteriaceae [2]. Human infection with shigatoxin producing E. coliO157:H7 (STECH O157) is relatively rare but the consequences could be serious, especially in the immune compromised such as the young and the elderly [3]. The outcome associated with STEC O157infection includes: Diarrhea, intense abdominal pain, hemorrhagic colitis, hemorrhagic uremic syndrome (HUS), kidney failure and eventual death [4]. The infection could be transmitted directly or indirectly through fecal-oral means with organism infecting its victim through braided skin, human or animal feces, contaminated food, water or soil. Out breaks has been associated with poor hygienic measures during slaughter, evisceration and processing of beef. The detection of E.coli O157:H7 is an indicator of fecal contamination and implies presence of other dangerous pathogens which can compromise the wellbeing of consumers [5] E. coli O157:H7 was widely distributed in North America, along with other serotypes such as STECO145, O26, O111 and O103, but studies have inculcated the organism to be found in processed beef and chickens egg in Africa and some other parts of the world [2]. Healthy ruminants especially feedlot cattle harbor this organisms in their lower gastrointestinal tract hence, constituting major reservoir of this organisms. Chickens and cattles, therefore, shed these organisms in their feces, thus, disseminating this deadly disease to the environment. Other well-known vectors for the transmission of E. coli O157O:H7 includes houseflies and formites [4]. Some factors are responsible for the pathogenicity of these organisms includes: Season of the year because fecal shedding rates is well known to occur during the summer and early rainfall. Also the age of chicken has shown that there is less shedding of the organisms in chicken of slaughter age than younger chicken [3]. The somatic antigen of E. coli O157:H7 is known to produce potent toxins which are shiga-like in nature and their distribution is based on the seasonal variability especially in healthy chicken which serves as apparent reservoirs of these organisms. The organisms could be discharged in meat, offal, milk and dairy products or contaminated water apple drinks, vegetables and bovine manure [3].

The risk of transmission of E. coli O157:H7 to man and animals has increased overtime. The fact that low infections dose of the organism as low as ten could trigger serious infection is a signal for more researches to be conducted on this disease. This, coupled with the short incubation period of the bacterium could further exacerbate in the disease especially in the elderly and immune-compromised young individuals below five years of age. The risk of E. coli O157:H7 from food animals has not been paid much attention in developing countries [6]. There is also paucity of information regarding the epidemiology of E. coli O157:H7 in developing countries. Animals are commercially slaughtered and dressed in unhygienic conditions which compromise microbiological quality and safety of meat obtained from the animals. This consequently risks the health of the consumers. 

To the best of my knowledge, there is limited public health surveillance data which characterizes E. coli isolates from chickens presented for slaughter in Fune abattoir, Nigeria. This research is the first work to be conducted in the study area in order to phenotypically characterize and identify E. coli from cloacal swabs of chickens presented for slaughter using standard bacteriological methods. This study serves as a catalyst for the need to the promotion of surveillance programs to identify sources of pathogenic E. coli from non-human origin.

Study area

Fune is a Local government area of Yobe state Nigeria. Its headquarter is Damagum town in the southwest of the are on the A3 highway at 11o40’39”N 11o20’04’E. It has an area of 4,948 km2 and a population of 300,760 at the 2006 census and most of which agrarians. The postal code of the area is 622 [7]. In 1987, the 8,000-year-old Dufuna canoe was discovered in fune near the village of Komadugu Gana River. The local has two constituencies; Jajere constituency and Damagum constituency. The populace tribes includes; Fulani, kare-kare, Ngizim and kanuri.

Sample Size Determination

The sample size is determined by using thrush field sample size calculation formulae:

Z=N/ (1+Ne2)

Where,

• N=population size= 3500

• e=0.05 at confidence level of 95%

• Thus, N=1000/(1+3500×0.0025)=114.25

• Approximately =114 [8].

Therefore a number of one hundred and fifty samples were collected from apparently healthy chickens in Yobe state to increase precision of the study.

Experimental Design 

A total of 150 samples (cloacal swab) were obtained from chickens from three different locations in Fune LGA. A sterile swab was inserted into the cloaca of the chicken and then turn slowly to take test fluid sample. The swabs were kept in a sterile tube containing 10ml of peptone water (transport/ pre enrichment medium), arranged in Ice Park cooler and transported immediately to Microbiology Laboratory, University of Maiduguri for analysis. The samples were collected in a period of 3 weeks. The locations for the samples collection sites are designated as follows: Damagum ward designated as Area D, Jajare as Area J, and Ngelzarma as Area N. 

Sampling 

Convenient sampling of 150 chickens was collected in three batches of chickens presented for slaughter from three different chicken slaughterhouses (chicken abattoir). Each Butcher, before taking the samples was interviewed orally to obtain information on sex of each chicken, and was told the type of research that will be done.

Collection of Cloacal Sample

The swabs samples were carefully obtained, taking care to avoid contamination from the outside of the cloaca, a sterile swab was inserted into the cloaca of the chicken and then rotator moved to take test fluid sample. Until delivery to the laboratory, the swab samples were collected unto sterile tube containing 10ml of peptone water (transport/ pre enrichment medium). The swab samples were kept refrigerated (in an Ice Park cooler) and transported to the laboratory.

Preparation of Culture Media     

All media were prepared based manufacturer’s instructions and the prepared media were then stored at 4°C for use during the cultivation. The specified media used for this research includes; peptone water, MacConkey agar, eosin methylene blue agar, nutrient broth medium, methyl-red medium and solution, triple sugar ion agar, Simmon’s citrate agar, and urea agar (Figure 1).

Figure 1: Map of Fune, Yobe State, Nigeria

The northeasterly line of equal latitude passes through the area including 11o30’00oN 11o30’00’E.

Culture and Isolation of E. coli

The processing table was first disinfected with alcohol (Ethanol). Each sample was immediately inoculated unto peptone water on arrival to the laboratory in other to propagate. This was sub cultured unto prepared MacConkey medium using a sterile wire loop and labeled accordingly and thereafter incubated at 37oC for 24hours to allow the bacterial growth. The presumptive positive E. coli isolates shows pink colonies (Lactose fermenting colonies) which were picked after 24 hours of growth and then sub cultured unto Eosin methylene blue medium and incubated at 37oC for 24 hours. The presumptive positive isolates appeared greenish metallic sheen on Eosin methylene blue medium were kept on nutrient agar slant and stored for future use (Figure 3).

Figure 3: Showing E. coli Colonies (Green metallic sheen with blackish center) on Eosin Methylene Blue Medium, this Shows a Clear Characteristics of E. coli Organism

The presumptive isolates were subjected to; gram staining, oxidase test, catalase test, and biochemical tests such as; indole, triple sugar ion, methyl red test, urease test, and citrate utilization test.

Preliminary Identification Test

Gram Staining: Gram staining method is most frequently used in diagnostic bacteriology. Clean slides with heat fixed smears were placed on a staining tray, the smears were flooded with crystal violet gently and let stand for 1 minute, the slides were tilted slightly and gently rinsed with tap water or distilled water using a wash bottle, the smears were flooded with lugals/Gram's iodine and let stand for 1 minute, the slide were tilted slightly and gently rinsed with tap water or distilled water using a wash bottle. The smears were appeared as purple as circle on the slide. Decolorized using 95% ethyl alcohol. The slides were tilted and applied alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Careful not to over-decolorize, the slides were rinsed immediately and flooded gently with safranin to counterstain and let stand for 45seconds. The slide were tilted slightly and gently rinsed with tap water using a wash bottle and blot dry slide bibulous paper. Finally the smeared slides were viewed using a microscope under oil immersion at 100x Magnification [9]. E. coli organisms are gram negative. Thus, they appeared pinkish/ red [9] (Figure 2).

Figure 2: Showing E.coli Colonies (Pinkish) on MacConkey Medium which Indicates that they are Lactose Fermenters

Catalase Production Test 

The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. This principle is used for detection of catalase enzyme in a bacterial isolate. A loopful of 10% hydrogen peroxide was put on colonies of the test organism on nutrient agar. Alternatively, a few colonies of the organism were picked up with platinum loop from nutrient agar slants and dipped in a drop of 10% hydrogen peroxide on a clean slide. The production gas bubbles from the culture, indicates a positive reaction. A false positive result may be obtained if the growth is picked up from the medium containing catalase e.g blood agar or if an iron wire loop is used [9].

Oxidase Test 

This test depends on the presence, in bacteria, of certain oxidase that catalyze the oxidation of reduced tetramethyl-p-phenylene-diamine dihydrochloride (oxidase reagent) by molecular oxygen. A drop of freshly prepared 1% solution of oxidase reagent was putted on a piece of filter paper. The a few colonies of the test organism were rubbed on it. Oxidase positive isolates, produced a deep purple colour within 10 seconds. Alternatively, oxidase reagent will be poured over the colonies of the test organism on culture plate. The colonies of oxidase positive rapidly develop a deep purple colour [9].

Complete Biochemical Characterization of the Presumptive Isolates

The presumptive isolates stored on nutrient agar slants were subjected to biochemical tests which were conventionally carried out on the isolates.

Indole Test 

Two to three colonies of E. coliisolates were inoculated in Bijou bottles containing peptone water using a wire loop and incubated for 24 hours. After 24 hours of incubation two to three drops of Kovac’s reagent were added to the medium and shaken. Formation of a pink layer was recorded as a positive result [10].

Citrate Utilization Test 

Using a straight sterile wire a colony of the test isolates were picked and streaked on Simmon’s citrate agar slope and stabbed to the butt before incubating at 37oC for 24 hours. Positive citrate test was indicated with a bright blue colour in the medium. No colour change indicated negative test [10]. 

Methyl-Red (MR) Test 

Two to three colonies of isolates were inoculated into methyl-red Voges proskauer (MR-VP) broth and incubated for 48 hours. Five to seven drops of methyl red solution were added to the broth and shaken. It was recorded positive since there was formation of a pink-red product within 5-15 minutes [10].

Urease Test 

Using a sterile wire loop, colonies of the isolates was picked and streaked on the urease slope and incubated at 37oC for 24 hours. A red pink colour change indicated positive test [10] (Figure 4).

Figure 4: Showing the Results from Biochemical Test from Right to Left; Urease (-ve), Citrate Utilization (-ve), Indole (+ve), and Triple Sugar Ion (+ve) Tests

Figure 5: Showing the Antimicrobial Sensitivity Test Result where most of the Organisms Show Some Resistance to Some Antibiotics by Producing Zone of Inhibition to Those Antibiotics

A total of 150 cloacal swab samples (101 males, 49 female chickens, 75 local and exotic each) were randomly sampled for the isolation of E. coli. The result show that 12 (25%) E. coli was isolated from female chickens, 40 (40%) was isolated from adult male chickens and 32 (42.67%) from local chickens while 20 (26.67%) was isolated from exotic chickens. The overall isolation rate was 52 (35%) (Table 1-2).

Biochemical Test

All the 52 positive isolates showed positive for Indole, Methyl red and Triple sugar ion tests. While negative result for Citrate, Catalase and Urease tests.

Antimicrobial Susceptibility Testing of the E. coli Isolates

The isolated E. coli from chickens (local and exotic) were subjected to in vitro antimicrobial susceptibility testing by the modified Bauer-Kirby method. The result showed that the isolates from exotic chickens aresusceptible to Ciprofloxacin 1 (5%) but were resistant to Cefixime 1 (5%), Cefuroxime 1 (5%) and Augmentin 1 (5%). Whereas, isolates from local chickens are susceptible to Ciprofloxacin 10 (31.3%), Ofloxacin 6 (18.8%), Gentamicin 8 (25%), Nitrofurantion 3 (9.4%), and were intermediate to Ceftazidime 1 (3.1%), Cefixime 1 (3.1%) and Cefuroxime 1 (3.1%), but were resistance to Cefixime 1 (3.1%) and Ceftazidime 1 (3.1%). Gentamicin had the widest zone of inhibition of (17mm).

Exotic chickens showed the highest resistance level of (15%) to commonly used antibiotics. Among all the chickens samples 16 (30.77%) of the positive isolates are susceptible to Ciprofloxacin, 4 (7.69%) to Nitrofurantion, 10 (19.23%) to Ofloxacin, and 12 (23.07%) to Gentamicin, while 2 (3.85%) were intermediate to Ceftazidime, 1 (1.92%) to Cefuroxime, 1 (1.92%) to Cefixime, and 1 (1.92%) to Augmentin. Whereas, 1 (1.92%) was resistance to Ceftazidime, 1 (9.2%) to Cefuroxime, 2 (3.85%) to Cefixime and 1 (1.92%) to Augmentin (Table 4-6).

Table 1: Distribution of E. coli Isolated From Chickens Presented For Slaughter in Fune LGA Based on Sex

Table 2: Distribution of E. coli Isolated From Chickens Presented For Slaughter in Fune LGA Based on Chickens Type

Table 3: Biochemical Reactions of Presumptive E. coli Isolates from Chickens Presented For Slaughter

Table 4: Antimicrobial Susceptibility Studies of E. coli Isolates from Chickens to Commonly Used Antimicrobial Agents

CAZ- Ceftazidime, CRX- Cefuroxime, GEN- Gentamicin, CXM- Cefixime, OFL-Ofloxacin, AUG- Augmentin, NIT- Nitrofurantion, CPR- Ciprofloxacin

Table 5: Antimicrobial Susceptibility Studies of E. coli Isolates from Local Chickens to Commonly Use Antimicrobial Agents

CAZ- Ceftazidime, CRX- Cefuroxime, GEN- Gentamicin, CXM- Cefixime, OFL-Ofloxacin, AUG- Augmentin, NIT- Nitrofurantion, CPR- Ciprofloxacin

Table 6: Antimicrobial Susceptibility Studies of E. coli Isolates from Exotic Chickens to Commonly Used Antimicrobial Agents

CAZ- Ceftazidime, CRX- Cefuroxime, GEN- Gentamicin, CXM- Cefixime, OFL-Ofloxacin, AUG- Augmentin, NIT- Nitrofurantion, CPR- Ciprofloxacin

This study was carried out to isolate and identify Escherichia coli at from chickens in Fune LGA, Nigeria. A total of 150 cloacal swab samples were collected from local and exotic chickens, where 52 showed positive for E. coli organisms. The isolation rate of E. coli in this study area is 34.66%. Escherichia coli occur aberrantly in most tissues or organs of the body (e.g. gastro intestinal tract, respiratory tract, urogenital tract) in man and animals [12]. Escherichia coli have been isolated in large numbers from faeces of ruminants and even humans [13-14]. The organism is implicated in causing several diseases in animals and man, with ruminant playing an important aspect in the epidemiology of the infections. Zoonotic E. coli (0157:H7) has been implicated in human outbreaks of haemorrhagic colitis, neonatal diarrhea and haemorrhagic uraemic syndrome [15]. Meat and meat product from animals can be contaminated by E. coli and transmitted to man via consumption of undercooked meat and poor hygienic practice (Table 3).

Over the past 60 years, antimicrobial have been used in a variety of setting including human medicine, veterinary medicine, plant agriculture and even cosmetics and antibacterial household products. All of these uses of antimicrobials potentially contribute to the emergence and spread of antimicrobial resistance but antimicrobial use in animals, particularly in food animals has recently come under particular scrutiny. In large part, concern stems from evidence for direct transfer of resistant pathogens from animals to humans through the food supply.

The result of the in vitro antimicrobial susceptibility study show that the Escherichia coli from chickens were susceptible to Ciprofloxacin, Ofloxacin and Gentamicin but were intermediate to Nitrofurantion and as well resistance to Augmentin, Cefixime, Ceftazidime and Cefuroxime. Generally, the organisms were susceptible to the flouroquinolones and this is in agreement with the report by Moses [13], and Ameh et al. [14]. Flouroquinolones are newer drugs with mode of action central on inhibition of DNA replication which stops the multiplication of the bacterial cells and are relatively expensive and therefore, are less available for abuse. The flouroquinolones (Ciprofloxacin, Ofloxacin and Gentamicin) had the widest zone of inhibition and are inversely related to minimum inhibitory concentration (MIC) values and this is in agreement with earlier report by Ameh et al. [14]. Resistance to Augmentin, Cefixime, Ceftazidime and Cefuroxime is in agreement with those of other workers [12-14]. The high incidence of drug resistance of Escherichia coli isolates as observed in this study may suggest that there was a wide spread transfer of resistance plasmid in the past [16] because resistance transfer in E. coli and other enterobacteriacae is often embolic and plasmid mediated. To generate baseline data to be used in future risk assessment of antimicrobial resistance, a number of surveillance systems on the local, continental and global scale is needed.

Base on the result of this study, it can be concluded that there is a intermediate distribution of the bacteria (E. coli) among chickens presented for slaughter in Fune Local Gov’t which may serve as a reservoirs of E. coli for transmission through their meat and by-products especially when slaughter conditions are not hygienic enough. This study has also shown the presence of antibiotic-resistant of E. coli from sampled chickens which could be as a result of acquisition of these antibiotic resistant bacteria by contact with carriers or ingestion of food (meat) and water contaminated by faecal droppings of other animals with previous expose to antibiotic. The study has established the presence of antibiotic-resistant bacteria. From the study, Ciprofloxacin, Ofloxacin, and Gentamicin had well activity against Escherichia coli and may be drugs of choice for the treatment of infections caused by E. coli but high resistance were observed to Cefixime, Cefuroxime, Ceftazidime and Augmentin where, exotic chickens showed the highest resistance level of (15%) to the commonly used antibiotics.

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