Study sites and sample collection
In this study, we collected raw meat samples from three slaughter sites in the Greater Accra region of Ghana. The sites consisted of a public slaughterhouse (PS), a slaughter slab (SS) and a privately-owned slaughterhouse (PO). Livestock such as cattle, sheep and goats were slaughtered daily at all three sites. Butchers operating at these slaughter sites obtained their livestock mainly from the three northern regions of Ghana. All three sites were points of meat sale and had the basic modules of production, which included a slaughter floor and lairage/holding pen. All parts of carcasses are sold as meat in large cuts and small cuts at these sites. The daily throughputs of livestock slaughtered at these sites ranged from 30-150 for large stock (cattle) and 20-300 for small stock (sheep and goats). Livestock at each site were selected randomly on predetermined sampling days. A total of 205 surface swab samples were collected from beef (n=81), chevon (n=108) and mutton (n=16) at the three slaughter sites (PS=63, SS=79, PO=63). Sample collection was done immediately after carcass dressing with a sterile cotton swab and a sterile template of size 100 cm2 for cattle and 25 cm2 for goat and sheep carcasses. For each beef carcass, surface swabs were collected from the thigh, brisket and flank and pooled into a single tube containing 10ml of Buffered Peptone Water (BPW). Swabs from goat and sheep carcasses was obtained from thigh, brisket and mid-loin (12). All samples were transported to the laboratory within 2 hours of sample collection for processing.
Enumeration of bacteria
Total plate counts and total coliform counts were performed for all samples using the pour plate method. Serial dilutions of ten-fold units of each sample were plated on Plate Count Agar (Oxoid) and incubated for 48 hours at 30°C. Following incubation, plates with colonies ranging from 30-300 were counted and expressed in CFU/cm2. The total coliform count was performed in the same way using Brilliance E. coli/ Coliform selective agar (Oxoid). The results of both counts were interpreted using guidelines set by the Ghana Standards Authority and the European Commission Regulation on the microbial criteria for food stuffs (13,14). Limits to total coliform counts were not specified in these documents hence limits set for Enterobacteriaceae counts in both documents were adopted for total coliform counts.
Identification of E. coli in meat
To isolate E. coli, samples were pre-enriched in Brain Heart Infusion (BHI) at 37°C for a period of 24 hours. Ten (10µl) microliters of each sample were then plated on MacConkey agar and incubated overnight at 37°C. We identified E. coli by colonial morphology and confirmed the isolates using Matrix Assisted Laser Desorption/Ionization -Time of Flight Mass Spectrometry (MALDI-TOF MS). Colonies from fresh overnight cultures were spotted on the MALDI-TOF MS target plate. One (1) µl of formic acid was then added and allowed to dry for 15 min. One (1) µl of matrix preparation was placed on each sample and left to dry for a further 15 minutes. MALDI-TOF MS was then conducted and ionization peaks (spectra) generated were matched against the integrated reference library of the MALDI system for confirmation of species of bacteria.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was done by the Kirby Bauer disk diffusion method using the following antimicrobial agents (Oxoid Ltd., Hampshire, UK): ampicillin (10ug), tetracycline (30ug), cefuroxime (30ug), cefotaxime (5ug), ceftriaxone (30ug), amikacin (30ug), gentamycin (10ug), chloramphenicol (30ug), ciprofloxacin (5ug), sulphamethoxazole trimethoprim (25ug) and meropenem (10ug). The procedure consisted of the preparation of a standard inoculum (0.5 McFarland) and inoculation on Mueller Hinton (MH) agar plates for 18 hours at 35°C (15). Interpretation of zone sizes was done using the EUCAST guideline (16). E. coli ATCC 25922 was used to monitor the performance of the test. In this study, we defined multidrug resistance (MDR) as resistance to three or more classes of antimicrobial agents.
Phenotypic and genotypic detection of ESBL production
The combination disk method was used to identify ESBL producing E. coli. Briefly, ceftazidime disks and ceftazidime combined with clavulanic acid disks were positioned 30 mm apart on the inoculated plates, and incubated overnight at a temperature of 37°C. Isolates were classified as ESBL positive if the difference between the inhibition zone diameter of ceftazidime combined with clavulanic acid and diameter of the inhibition zone for the ceftazidime-only disk was ≥5mm (17). Cefotaxime disks and cefotaxime combined with clavulanic acid disks were used concurrently with ceftazidime for confirmation of ESBL production. DNA was extracted from presumptive colonies for the genotypic detection of ESBL production.
Bacterial DNA extraction consisted of placing pure colonies of overnight growth in 1ml of distilled water. The mixture was boiled for 10 minutes and centrifuged for 5 minutes, at 1000 rotations per minute. Using the supernatant as a DNA template, PCR was done to detect ESBL resistance genes (blaTEM, blaSHV, and blaCTX-M). The final volume of 25µl contained 2µl DNA template, 10mM of each primer (Table 1) (18), PCR grade water (10ul) and multiplex PCR master mix (13ul) (QIAGEN). Multiplex PCR was carried out in a thermal cycler with the following cycling conditions: 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes, and a final extension lap at 72°C for 10 minutes.
Statistical analysis
Descriptive statistics were performed on carriage rates of E. coli and their resistance patterns. The Chi-square test was used to assess differences in the proportion of E .coli detected in raw meat between the three slaughter sites. A Kruskal-Wallis test was performed to determine if there were significant differences in microbial counts in raw meat among the slaughter sites. Statistical analyses were performed at 95% confidence level using STATA version 15.0.