2.1 Study Area
The study was conducted in Sokoto metropolis, Nigeria. Sokoto metropolis is made up of four local government areas, namely, Sokoto North, Sokoto South, Wamakko, and Dange-Shuni. The state is located at latitude 13∘ N and between longitudes 4∘ 8’ E and 6∘ 54’ E in North-western Nigeria. The State covers an area of approximately 56,000 square kilometres [13]. The State shares a border with the Niger Republic to the north, Kebbi State to the south, and Zamfara State to the east. Based on the 2006 census, Sokoto metropolis was estimated to have a population of about 4,344,399. The State is ranked second in livestock population with about 3 million cattle, 4 million goats, 3.85 million sheep, 0.8 million camels, and 1 million poultry (Sokoto metropolis Investment Promotion Committee (SSIPC).
2.3 Sample Size Determination
The minimum sample size for this study was determined by the formula
n = t2x pexp(1-pexp)/ d2 [14]
Where n = sample size, t2 = the score for a given interval, which is 1.96(S.E.) at 95% confidence interval, pexp = known or estimated prevalence, d2 = and precision at 0.05.
The sample was calculated at a 12.1% expected prevalence [15], a 95% confidence interval, and a desired precision of 5%.
n = (1.96)2 × 0.08 × (1-0.08)/ (0.05)2,
n = 0.307328× (0.92)/ 0.0025 = 163.446704
Thus, n = 165
2.4 Study Design and Sample Collection
A cross-sectional study was conducted where carcass rinsates were collected from dressed broiler chickens at different slaughter points. A systematic random sampling technique was used to collect the rinsate from the nth chickens during standard daily processing. One hundred and sixty-five rinsates were obtained from 4 different poultry processing sites (slaughter slabs) in the metropolis. Methods described and adapted by De Cesare et al. [16] were used to collect the rinsates. Briefly, with a gloved hand, a completely dressed carcass was immersed in a sterile bag containing 30 mL of sterile distilled water. Subsequently, it was shaken vigorously, and the chicken was aseptically removed from the immersed distilled water. Sterile bags containing samples were adequately labelled and transported on ice packs and were immediately analysed at the Fleming laboratory, Veterinary Teaching Hospital, Usmanu Danfodiyo University, Sokoto.
2.5 Culture and Isolation of E. coli
The collected samples were aseptically inoculated (1ml) into buffered peptone water (Oxoid, UK) and incubated aerobically at 37°C for 24 hrs. Then, a loopful of the broth culture was streaked onto MacConkey agar (Oxoid, UK) and incubated aerobically at 37°C for 24–48 hours. The lactose fermenting (pink) colonies were inoculated onto Eosin Methylene Blue (EMB) agar (Oxoid, UK). Colonies with a green metallic sheen appearance were taken as presumptive E. coli and were transferred onto a slant bottle for further processing [17].
2.6 Biochemical Characterization of E. coli
Presumptive E. coli was subjected to biochemical identification using the Indole reaction, Methyl red test, Voges-Proskauer test, and Citrate utilisation test (IMViC), as described by Cheesbrough and Tawyabur et al. [18,19].
2.7 Molecular Confirmation of E. coli
The genomic DNA of E. coli was extracted as described by Salah et al. [20]. Briefly, 2–3 colonies of E. coli were transferred into 200µL of nuclease-free water and were boiled for 10 minutes to disrupt the cells and release the DNA, followed by centrifugation at 10,000 rpm for five minutes. The crude DNA extract supernatant was stored at -20°C until polymerase chain reaction (PCR) was used. Confirmation of E. coli was done by PCR amplification of the β-d-glucuronidase (uidA) gene using specific primers [21,22]. PCR amplification was done using the PCR reaction at a final volume of 25µl reaction mixture containing 12.5µl of ThermoFisher Master Mix, 0.5µl each of the forward and backward primers, 9.5µl of nuclease-free water (Invitrogen, Carlsbad, CA), and 2µl of DNA template. The amplification cycle consists of an initial denaturation of 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, annealing at 60°C for 30 s in 35 cycles, initial extension at 72°C for 30 s in 35 cycles and a final extension at 72°C for 5 min in 1 cycle in a T100 Thermal cycler (Bio-Rad Laboratories, Inc. USA). PCR products were separated on a 1.5% (w/v) agarose gel in Tris Borate EDTA buffer (pH 8.2), stained with ethidium bromide (10µg/ml). They were visualised using the GelDoc Go Imaging System (Bio-Rad Laboratories, Inc., USA).
2.8 Antibiotic Sensitivity Testing of Isolates
Confirmed E. coli isolates were tested for susceptibility to ten of the most common antimicrobials used in the field, which include Ampicillin (10µg), Gentamycin (10µg), Cefotaxime (30µg), Ceftazidime (10µg), Ciprofloxacin (5µg), Chloramphenicol (30µg), Nalidixic acid (30µg), Sulphonamide (300µg), Tetracycline (30µg), and Trimethoprim (5µg). Susceptibility testing was done using the Kirby–Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI, 2020). According to the guidelines, the antimicrobial susceptibility was based on the induced inhibition zones [23]. Inhibition zone data was entered into WHONET version 5.6, configured with the tested antimicrobials. Isolates were categorised as sensitive, intermediate, or resistant using clinical breakpoints and guidelines of CLSI (2020). Resistance to at least one agent in three or more antimicrobials of different categories was taken as multidrug resistance (MDR), according to Dadgostar (2019) and Magiorakos et al. (2012).
2.9 Molecular Detection of Resistance Genes
Isolates that were phenotypically multidrug resistant were subjected to PCR amplification of tetracycline resistance genes (tetA, tetB) [24], sulphonamide resistance genes (sul1, sul2) [25]; [26] and chloramphenicol resistance genes (catA1, catA2) [27]; [28] using specific primers.
Multiplex PCR was performed to amplify sul2 and tetA. The PCR was performed in a 25µl reaction volume consisting of 12.5µl of a master mix (Biolabs), 0.5µl of forward primers (IDT®), 0.5µl of reverse primers (IDT®), 6.5µl of nuclease energy-free water (Biolabs), and 4µl of DNA template. The PCR was performed using the DNA amplification method comprising an initial denaturation step at 94℃ for 5 minutes, 35 cycles of denaturation at 94℃ for 30 seconds, 35 cycles of annealing at 53℃ for 30 seconds, and an initial extension at 72℃ for 30 seconds in 35 cycles also and one final extension cycle at 72℃ for 5 minutes. The PCR products were loaded in a 1% agarose gel (Vivantis Incorp, USA), 1x TBE (Vivantis Incorp, USA) buffer (Tris 0.09M- borate 0.09 M-EDTA 0.02M) pre-stained with ethidium bromide (Biotium, Hayward, USA). Electrophoresis was carried out at 70 volts for 70 minutes (Bio-Rad Laboratories, Inc., USA). Molecular markers of 100bp (GeneDirex, Taiwan) were run in parallel with the DNA samples to indicate the size of PCR amplicons. Gel was visualised under a UV light transilluminator (Bio-Rad Laboratories, Inc., USA), and the images were taken.
Uniplex PCRs were performed to amplify sul1, tetB, catA1, and catA2. The PCRs were performed in a 25µl reaction volume consisting of 12.5µl of a master mix (Biolabs), 0.5µl of forward primers (IDT®), 0.5µl of reverse primers (IDT®), 9.5µl of nuclease energy-free water (Biolabs), and 2µl of DNA template. The PCRs were performed using DNA amplification methods that comprised temperature conditions, time, and cycles, as described elsewhere (include references).
2.10 Data and Statistical Analysis
Data was entered into Microsoft Excel 2016 and later exported to SPSS version 23 (IBM, USA) for inferential statistics. The chi-square test was used to check for any association between the prevalence of E. coli and slaughtering points. Cohen’s kappa statistic was used to assess the level of agreement between phenotypic resistance and the detection of sulphonamides, chloramphenicol, and tetracycline resistance genes. Cohen’s kappa is a statistical measure used to evaluate the level of harmony between two test protocols, assigning a kappa value between zero and one. Values ≤ 0 indicate no agreement; 0.01–0.20 none to slight; 0.21–0.40 fair; 0.41–0.60 moderate; 0.61–0.80 substantial; and values in the range of 0.81–1.00 indicate values with nearly perfect agreement (McHugh, 2012). A p-value < 0.05 was considered statistically significant at a 95% confidence interval