Extended-spectrum beta-lactamase-producing Escherichia coli in chickens from small-scale ( backyard ) poultry farms in Maiduguri , Nigeria

Aim: This study investigated the occurrence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in chickens from small-scale (backyard) commercial poultry farms in Maiduguri. Materials and Methods: A total of 96 cloacal swab samples were collected. This comprised of 24 samples each from broiler chicks, pullets, layers, and broilers (adults). The samples were examined for the presence of E. coli using conventional microbiological culture and biochemical tests. The pure E. coli isolates were screened for ESBL production by culturing onto BrillianceTM ESBL agar. Isolates that showed positive reactions with production of bluish or pinkish colonies were tested for susceptibilities against some selected beta-lactam antibiotics which include cefotaxime (30 μg), ceftriaxone (30 μg), cefpodoxime (10 μg), aztreonam (30 μg), and ceftazidime (30 μg). Isolates that exhibited resistance to any two or three of the antibiotics were selected and confirmed by combination disk diffusion method with ceftazidime (30 μg) and cefotaxime (30 μg) alone and in combination with clavulanic acid (30 μg/10 μg). Results: The total occurrence of E. coli was 67.6% (65/96) with the highest occurrence of 83.3% (20/24) from broiler chicks and least detection of 54.2% (13/24) from layers. Of this, 32.0% were ESBL-producing E. coli with the highest detection rate from layers (38.5%) and least occurrence from pullets (26.7%). Conclusion: This study revealed the presence of ESBL-producing E. coli in chickens from small-scale commercial poultry farms in Maiduguri, thus indicating that chickens may serve as important reservoirs for the transmission of antimicrobial resistant pathogens to humans through the food chain.


Introduction
The surge in the prevalence of antimicrobial resistance from foodborne pathogens and the natural environment is one of the most significant threats to animals and humans in the 21 st century [1][2][3][4].This is due to the narrowing down in the cache of antibiotics both in quantity and quality and due to the gap between antimicrobial resistance and development of antibiotics [5][6][7][8].Food-producing animals are among the most important reservoirs of antimicrobial resistance genes [1,9,10], due partly to the frequent and indiscriminate use of antimicrobial agents as feed additives for prophylaxis and in the treatment of gastrointestinal illness in animals [4,11], thus facilitating the shading of resistance genes and pathogenic foodborne bacteria into the environment [12].
The emergence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in humans and animals has gained global notoriety during the past decade.This comes along with a fiery concern of food animals serving as potential reservoirs of ESBL genes.Since ESBL are plasmid coded, their transfer from animals to humans cannot be overemphasized [13].In addition, some strains of E. coli have been observed to play a pivotal role in the spread and maintenance of ESBL genes [14,15].ESBL act by inactivating beta-lactam antibiotics such as penicillin and third-generation cephalosporins through hydrolysis of their beta-lactam ring.The enzymes are found majorly in Enterobacteriaceae, members of the normal gut flora causing opportunistic to severe bloodstream and urinary tract infections [16][17][18].
The poultry industry has assumed greater importance in improving employment opportunities and animal food production in Nigeria [19][20][21].Earlier reports showed that poultry production accounts for Available at www.onehealthjournal.org/Vol.5/4.pdf about 36.5% of sources of protein in Nigeria [22,23].However, the viability and growth of this sector is hampered by diseases which have consequently resulted in reduced output in terms of production [12].In many communities in Nigeria, small-scale poultry farms constitute a large portion of poultry farming activities, and previous studies have reported that this poses a major risk in the dissemination of multidrug-resistant pathogens in the environment [24].
In view of the increasing concerns about ESBLproducing E. coli in food animals and the possible risk that they pose to public health [25], this study was carried out to investigate the presence of ESBLproducing E. coli in small-scale poultry farms within Maiduguri.

Ethical approval and informed consent
The study design was approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine, University of Maiduguri.Also, informed consent from farmers was obtained for the inclusion of their farms in the study.

Sampling
A total of 96 cloacal swab samples consisting of 24 samples each from broiler chicks, broilers, layers, and pullets were collected and used in this study.The samples were collected from chickens on deep-litter systems in small-scale farms located at Lake Chad, Damboa Road, Mairi, and New GRA wards of Maiduguri.Cloacal swab samples were collected with the aid of pre-labeled sterile swab-sticks dipped in peptone and packed in polystyrene cooler containing ice-packs for immediate transportation to Bacteriology Research Laboratory, Department of Veterinary Microbiology, University of Maiduguri.

Bacterial isolates
The bottles containing cloacal swabs were incubated for 2 h at 37°C aerobically.After enrichment, the samples were then streaked onto freshly prepared eosin methylene blue (EMB; Oxoid, UK) agar and incubated aerobically at 37°C for 24h.E. coli colonies on EMB with the appearance of very dark and with a metallic green sheen were sub-cultured onto Nutrient Agar (Oxoid, UK) and incubated at 37°C for 24 h.The colonies were subjected to gram staining for cellular morphology and a series of biochemical tests according to Jang et al. (2004).Identified E. coli isolates were kept on nutrient agar slant (Oxoid, UK) at room temperature until used.

Antibiotic susceptibility test and confirmation of ESBL production
Pure E. coli isolates were screened for ESBL production by culturing onto a Brilliance ™ ESBL agar (Oxoid, UK) a chromogenic screening plate for the detection of ESBL-producing organisms.The plates were then incubated at 37°C for 24 h.ESBL-producing E. coli were identified by characteristic bluish or pinkish colonies.The ESBL-producing E. coli were tested for susceptibility using disk diffusion method as described by Bauer et al. [26].The ESBL-producing E. coli were tested against five beta-lactam antibiotics which included cefpodoxime (10 µg), ceftazidime (30 µg), aztreonam (30 µg), cefotaxime (30 µg), and ceftriaxone (30 µg).The diameters of inhibition zones were measured and interpreted as per the guidelines of the Clinical and Laboratory Standards Institute [27].All the isolates that showed resistance to any of the two or three cephalosporins were subjected to confirmation for ESBL production using combination disk diffusion method with ceftazidime (30 µg) and cefotaxime (30 µg) alone and in combination with clavulanic acid (30 µg/10 µg) (Oxoid, Basingstoke, UK).The E. coli isolates were phenotypically considered as ESBL producers when there is an increase of ≥5 mm in the size of inhibition zone for antimicrobial agents when tested along with clavulanic (ceftazidime-clavulanic acid or cefotaxime-clavulanic acid) in addition to the initial zones of inhibition when ceftazidime and cefotaxime were tested alone

Statistical analysis
The data obtained from this study were summarized and presented for descriptive purposes in the form of tables as percentages.Chi-square analysis was used to test for the level of statistical significance in the association of the occurrence of E. coli and ESBL-producing E. coli from chickens with respect to chicken types and age using GraphPad Instat3 (GraphPad Software, 2365 Northside Dr. Suite 560 San Diego, CA 92108).Relative risk and confidence interval of 95% were used for the determination of level of statistical significance and the values of p<0.05 was considered statistically significant.

Discussion
In this study, a total of 96 cloacal swab samples were collected from chickens in small-scale poultry farms in Maiduguri and screened for the presence of ESBL-producing E. coli.Total prevalence of 67.7% of E. coli was obtained with the highest occurrence of 83.3% in broiler chicks.This agrees with the findings of Kegode et al. [28] where a similar occurrence of E. coli from chickens was also reported.The finding of this study also showed that the occurrence of E. coli in chickens <4 weeks of age (72.9%) was higher compared to birds of age >4 weeks (Table -2).This finding is not in agreement with other previous studies where the authors reported 44% prevalence of E. coli in chicks [29].The high occurrence of E. coli in chicks from this study could be linked to a lack of good sanitary conditions observed in the farm environments during this work.It was noted from this study that most small-scale farmers entrust their farm management to individuals who have little or no formal education and experience in poultry farm practice and therefore give little attention to the hygiene of birds and the environments.Hence, creating a conducive atmosphere for bacterial growth and colonization.In addition, the high occurrence could also be attributed to sampling source and type of samples, and for the fact that E. coli is a normal gut flora, [30][31][32].
In this study, a total occurrence of 32.3% of ESBL-producing E. coli was observed in chickens.Similar prevalence of ESBL-producing E. coli in chickens was also reported in Zambia by Chishimba et al. [6].This finding is lower than the findings of previous studies [33][34][35], where higher occurrences of ESBL-producing E. coli were reported in chicken meat.It was also observed from this study that the highest occurrence of ESBL-producing E. coli was from laying birds (layers) (38.5%).Based on this, it is important to note that layers are normally kept for a longer period and therefore may have prolonged exposure to antibiotics for prophylaxis which might result in the selection of drug-resistant bacterial pathogens.However, since no statistically significant difference was observed (p>0.05) in the occurrence of the pathogen in chickens with respect to chicken-types, it implied that both broilers and layers are at risk of harboring the organism when raised under conditions that support the selection of antimicrobial resistant pathogens.The occurrence of ESBL-producing E. coli in broilers is higher than the findings of Shoaib et al. [32] where 7.76% occurrence rate was reported.Furthermore, results of analysis of the occurrence of ESBL-producing E. coli in chickens based on age showed no statistically significant difference on the occurrence of the organism (p>0.05) which implies that both age groups may be at risk of harboring the pathogens if managed under conditions that promote transmission and acquisition of the organism.

Conclusion
The study affirmed the presence of ESBLproducing E. coli in chickens from backyard poultry farms in the study area.These isolates also showed a high level of antibiotic resistance to third-generation cephalosporins and the monobactam, aztreonam.This is of serious public health significance since birds are reared in close proximity to human population and may disseminate these resistant pathogens in the environment and in-contact farm personnel.
Copyright:Kwoji, etal.This article is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
/15)as shown in Table-1.The result of this study also showed no statistically significant difference in the occurrence of ESBL-producing E. coli in chickens based on types and age (Table-3) (p>0.05)(Table-3).The presumptive ESBL-producing E. coli isolates were tested for susceptibility against

Table - 1
: Occurrence of E. coli and ESBL-producing E. coli in chickens from small-scale poultry farms in Maiduguri.Occurrence of E. coli in chickens from small-scale poultry farms in Maiduguri based on chicken types and age.

Table - 3
: Occurrence of ESBL-producing E. coli in chickens from small-scale poultry farms in Maiduguri based on chicken types and age.