4.2.1. Study design and study sites
A cross-sectional, multicentre study was conducted between March and October, 2016 in three slaughterhouses/markets in Cameroon and two abattoirs in South Africa, that were encoded for ethical reasons as SH001, SH002, SH003 and SH004 and SH005, respectively. All slaughterhouses were visited at different time points to allow better representativeness of the pig population.
In Cameroon, samples were randomly taken at the two biggest pig abattoirs (SH001 and SH002) of Yaoundé, where more than 80% of pigs are slaughtered and with annual productivity scale ranging from 25 000 to 45 000 pigs (12). Samples from an auxiliary abattoir (SH003)
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were additionally collected to ensure a geographically representative distribution since animals originating from surrounding farms of Yaoundé are typically underrepresented in these slaughterhouses. These slaughterhouse/markets were located in three geographically distinct districts of Yaoundé. In South Africa, samples were taken at two of the biggest and most productive abattoirs of the province of KwaZulu-Natal (SH004 and SH005), with annual productivity ranging from 120 000 to 150 000 pigs.
4.2.2. Ethical considerations
Ethical approvals from the Biomedical Research Ethics Committee (Ref. BE365/15) and Animal Research Ethics Committee (Ref. AREC/091/015D) of the University of KwaZulu- Natal as well as from the National Ethics Committee for Research in Human Health of Cameroon (Ref. 2016/01/684/CE/CNERSH/SP) were obtained prior the implementation of the study. Ministerial approvals from the Cameroonian Ministry of Livestock, Fisheries and Animal Industries (Ref. 061/L/MINEPIA/SG/DREPIA/CE) and Ministry of Scientific Research and Innovation (Ref. 015/MINRESI/B00/C00/C10/C14) were also obtained.
4.2.3. Sampling procedure and questionnaire a. Procedures for animal data
A randomized sampling method of apparently healthy and freshly slaughtered/stunned pigs was undertaken in both countries. Nasal (inner cavity of both anterior nares) and rectal swabs of pigs were collected using Amies swabs without charcoal (Copan Italia Spa, Brescia, Italia).
Overall, 864 swabs (432 nasal and 432 rectal) were collected from 432 pigs in Cameroon (n=216) and South Africa (n=216), with the number of specimens from each slaughterhouse (SH001, n=129; SH002, n=57; SH003, n=30; SH004, n=120; SH005, n=96) being proportionally calculated to be representative of the number of pigs slaughtered annually per site.
b. Procedures for human subjects
All abattoir workers older than 21 years old and willing to participate were included in the study after oral and written informed consent. Upon this step, participants were asked to complete a questionnaire addressing socio-demographic and clinical information, as well as other potential risk factors associated with ESBL-PE colonization and dissemination. Both anterior nares and hands (between fingers for each right and left hand) were collected with Amies media and all samples were processed within 4 h after collection.
4.2.4. Laboratory analysis
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For microbiological analysis, three individual pig samples were pooled per slaughterhouse and according to the gender, specimen type and area of breeding to yield 144 nasal and 144 rectal pools representing 432 original nasal and rectal samples respectively collected from 432 pigs.
A total of 288 swabs from the 144 nasal and 144 rectal pools constituted the final pig sample.
Pooled samples of pigs and human swabs were streaked onto an in-house screening MacConkey agar supplemented with 2 mg/L cefotaxime (MCA+CTX) and incubated for 18- 24 h at 37°C for ESBL-PE screening. Presumptive ESBL-PE were phenotypically confirmed with Vitek® 2 System (BioMérieux, Marcy l’Etoile, France).
a. ESBL detection, species identification and antimicrobial susceptibility testing Each colony growing on MCA+CTX and with a unique morphotype was screened for the production of ESBLs through the standard double disk synergy test (DDST), using cefotaxime and ceftazidime, alone and in association with clavulanic acid as recommended by the Clinical Laboratory and Standards Institute (CLSI). An increase in size of the inhibition zone of more than 5 mm in the presence of clavulanic acid was regarded as positive for ESBL production (13). Upon this two-step screening, a representative subset of isolates, underwent phenotypic identification via Vitek® 2 System (BioMérieuX, Marcy l’Etoile, France).
Using an 18-24 h fresh culture, 1 to 2 colonies were mixed with 3.70 ml of sterile saline solution, resulting into a 0.5 turbidity on the McFarland scale. The minimum inhibitory concentrations (MIC) of ampicillin, amoxicillin + clavulanic acid, cefuroxime, cefuroxime acetyl, cefoxitin, cefotaxime, ceftazidime, cefepime, ertapenem, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, tigecycline, nitrofurantoin, piperacillin/tazobactam, colistin and trimethoprim-sulfamethoxazole, were determined by broth microdilution method using Vitek® 2 System (BioMérieuX, Marcy l’Etoile, France) and Vitek® 2 Gram Negative Susceptibility card (AST-N255) (BioMérieux, Marcy l’Etoile, France). The results were interpreted according to the CLSI guidelines (13) with the exception of colistin, amoxicillin + clavulanic acid, piperacillin/tazobactam and amikacin that were based on EUCAST breakpoints (14) with E. coli ATCC 25922 being used as the control.
b. Genomic Extraction
Genomic DNA was extracted from a subset of ESBL-PE strains selected on the basis of their antimicrobial resistance profiles. Samples were cultured in 3 ml of Tryptone soya broth with moderate shaking for 18 h at 37oC in normal atmosphere. After incubation 1.5 ml of broth was centrifuged at 12,000 g for 10 min and the pellet was prepared for genetic analysis using the Thermo Scientific® GeneJet Genomic DNA purification kit (Thermo Fisher Scientific, South
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Africa) according to the manufacturer’s instructions. DNA templates were stored at -20°C until used.
c. Genotypic relatedness determination of ESBL-producing Escherichia coli strains Genomic DNA was extracted using the Thermo Scientific® GeneJet Genomic DNA purification kit (Thermo Fisher Scientific, South Africa) according to the manufacturer’s instructions. ERIC-PCR was performed with primers ERIC 1 5’-ATG TAA GCT CCT GGG GAT TCA C-3’ and ERIC2 5’-AAG TAA GTG ACT GGG GTG AGC G-3’ (8). Reactions were carried out in a 10 µl final solution containing 0.1 µl of each primer (100 μM), 5 µl DreamTaq Green Polymerase Master Mix 2× (Thermo Fisher Scientific, South Africa), 2.8 µl nuclease free water and 2 µl DNA template and run in an Applied Biosystems 2720 programmable thermal cycler (Thermo Fisher Scientific, South Africa) with the following protocol: initial denaturation at 94°C for 3 min, 30 cycles consisting of a denaturation step at 94°C for 30 s, annealing at 50°C for 1 min, extension at 65°C for 8 min, a final extension step at 65°C for 16 min and final storage at 4°C. ERIC profiles were digitized for analysis using Bionumerics software (version 7.6, Applied Maths, TX, USA). The similarity between each strain was determined from the homology matrix using Dice coefficient and dendrograms constructed using the algorithm Unweighted Pair-Group Method (UPGMA).
d. Data analysis
Data was coded and entered into Excel spreadsheet (Microsoft Office 2016) and Epi Info (version 7.2, CDC, Atlanta, GA, USA), and analysed STATA (version 14.0, STATA Corporation, TX, USA). A data set was created for individual human results and, aggregated animal and abattoir data. Abattoirs were classified as ESBL-positive if an ESBL-PE was detected from at least one pooled sample (nasal or rectal). Likewise, each human was categorized as carrier or non-carrier, with carrier being defined as having ESBL-PE in at least one site (nose or hand).