3. Discussion
Enterobacteriaceae and especially ESBL-PE, were acknowledged as critical priority antibiotic-resistant bacteria (ARB) by the WHO and their emergence at the animal-human-environment interface presents a to serious and multifaceted public health concern globally [
3]. This study investigated the carriage, risk factors, antibiotic resistance profiles and genetic relatedness of ESBL-PE isolated from apparently healthy pigs and occupationally exposed workers in Cameroon and South Africa.
The overall prevalence of human ESBL-PE carriage was 50% in hand and 45.75% in nasal samples. Comparable data was reported by Magoue et al. (2013) in Cameroon, where the prevalence of ESBL-PE faecal carriage was 45% in outpatients in the region of Adamaoua [
6]. Our findings are nevertheless higher than that described by Dohmen et al. (2015) where a 27% prevalence of ESBL-PE carriage in faecal samples of people with daily exposure to pigs in Netherlands was described [
7].
Our results are in contrast to a study of Fisher et al., (2016), where none of the 66.7%
Enterobacteriaceae detected in the nares of participants were ESBL producers and where the authors concluded that nares were a negligible reservoir for colonization of ESBL-PE in pig’s exposed workers [
8]. Our finding shows that the prevalence of ESBL-PE carriage in nasal samples substantially increased (8.33 vs. 91.67%;
p < 0.001) and was statistically significantly correlated with their carriage on hand (OR 39.11; 95% CI 2.02–755.72;
p = 0.015). In addition, nasal ESBL-PE carriage was associated with inappropriate handwashing with high statistical significance (OR 4.71; 95% CI 2.28–9.70;
p < 0.001). This suggests, that nares might likely become reservoir of ESBL-PE when limited hygienic conditions prevail and biosecurity measures are not adequately implemented. It further reveals that, as with the transmission of nosocomial infections in hospital settings, hands constitute important vectors of ABR transmission in the food production industry and may not only drive the transfer from person-to-person but also the contamination of food products intended for the end consumer. Nasal ESBL-PE carriage reported herein might also be ascribed to airborne contamination as recently reported by Dohmen et al., (2017) who revealed that human CTX-M-gr1 carriage was statistically associated with presence of CTX-M-gr1 in dust (OR = 3.5, 95% CI = 0.6–20.9) and that inhalation of air might constitute another transmission route of ESBL-PE in the food chain [
9].
The difference in the prevalence of ESBL-PE carriage in humans in both countries could be explained by the fact that South Africa has existing abattoir regulations in place and South African abattoirs were compliant with international food safety standard ISO 22000 and Hazard Analysis Critical Control Points (HACCP) plans. In Cameroon, slaughterhouse/markets were principally low-grade, lacking in basic amenities, with sub-optimal sanitary conditions and limited or non-existent biosecurity measures. The Food and Agriculture Organization for the United Nations (FAO) report on abattoir facilities in Central African countries including Cameroon, already underlined the gaps in term of biosecurity measures in these settings [
10]. Our findings, therefore, reinforce the importance of and the need to implement strict biosecurity procedures as when effective prevention and containment measures are implemented, the risk of ABR dissemination is reduced.
The overall prevalence of ESBL-PE in pigs was 71% and 75% in rectal and nasal pooled samples, respectively. The results are consistent with that reported by Le et al., (2015) in food animals and products in Vietnam where a 68.4% prevalence of ESBL-producing
E. coli was described [
11]. They are however lower than that reported in pig farms in Germany, where 88.2% of ESBL-producing
E. coli was detected [
12] and higher than that reported in two other studies with prevalence ranging from 8.6 to 63.4% in food animals and food products in Netherlands, [
13] and 8.4% in cattle in Switzerland [
2].
The high rate of ESBL-PE carriage detected in both nasal and rectal samples in Cameroon may suggest that ESBL-PE are consistently widespread in food animals in Cameroon, disseminate in the farm-to-plate continuum and represent a grave public health threat in the country. Similarly, the ESBL-PE prevalence detected in pigs in South Africa is not surprising, especially because the use of antibiotics as growth promoter agents is legally allowed in the country [
14]. These findings reveal gaps in the current state of knowledge about antibiotic use and ABR in food animals and suggests that the debate about ABR-related consequences in the farm-to-plate continuum is neglected in Cameroon and South Africa and should be more seriously considered in these countries. Additionally, our study revealed a high frequency (95%) of ESBL-producing
E. coli, emphasizing the relevance of this indicator bacteria as a serious public health issue.
ERIC analysis demonstrated relative associations amongst human and animal isolates within and across countries. Some strains isolated in humans were highly related to those detected from pigs at similar or dissimilar abattoirs suggesting that the occurrence of ESBL-PE in humans may have an animal origin or vice-versa and that these bacteria may spread to humans via the food chain, allowing their dissemination to the global population. Although not providing evidence on the transmission dynamics of ESBL-PE, our results nevertheless show an epidemiological link amongst isolates from humans and animals.
Hospitalization, antibiotic use and contact with (food) animals are known risk factors for human ESBL-PE carriage [
15]. Twenty-one abattoir workers or their family members had been admitted to a hospital within the year of the sampling. Of these, 39.29% evidenced nasal ESBL-PE carriage and 71.43% hand ESBL-PE colonization (
Table 1). Likewise, the majority of workers who had used antibiotics within the month of the sampling were colonized by ESBL-PE in nares (55.26%) and hands (71.05%) (
Table 1).
There are certain limitations to consider in this cross-sectional study. First, the duration of ESBL-PE carriage was not investigated and there was no apparent relationship between human ESBL-PE carriage and contact with ESBL-PE colonized pigs (
Table 6). Secondly, in contrast, a clear association was established between contact with other (food) animals, mainly poultry and human ESBL-PE colonization, with high statistical significance (
Table 6), suggesting that further work should be undertaken in high risk populations and other food animals such as poultry in order to expand our understanding on the public health impact of the likely zoonotic transmission of ESBL-PE through the farm-to-plate continuum. Thirdly, the small human sample size precluded any direct conclusions on the prevalence of antibiotic resistance among abattoir workers. Finally, the molecular analyses were only carried out on a representative sub-sample and not all isolates due to financial constraints. Comprehensive molecular analysis would have certainly allowed better understanding of the genetic exchanges and evolution that are likely to occur within and between bacteria in this continuum.
To the best of our knowledge, this is the first report of ESBL-PE in animals and humans in both Cameroon and South Africa taking food safety perspective. The high prevalence of ESBL-PE found in pigs in both countries as well as in humans in Cameroon highlights the food safety issue associated with their presence in the farm-to-plate continuum. It demonstrates the urgent need to implement multi-sectorial, multi-faceted and sustainable collaboration and activities among all stakeholders involved in this continuum in order to reduce the prevalence and contain the dissemination of ESBL-PE and ABR in these countries.
4. Methods
4.1. Study Design and Study Sites
From March to October 2016, a multicentre study was conducted in five abattoirs in Cameroon (n = 3) and South Africa (n = 2). All abattoirs were coded for ethical reasons as SH001, SH002, SH003, SH004 and SH005. They were visited thrice at different time points to allow a representative sample.
4.2. Ethical Considerations
Prior to the implementation of the study, ethical approvals were obtained from the National Ethics Committee for Research in Human Health of Cameroon (Ref. 2016/01/684/CE/CNERSH/SP), Biomedical Research Ethics Committee (Ref. BE365/15) and Animal Research Ethics Committee (Ref. AREC/091/015D) of the University of KwaZulu-Natal. In addition, ministerial approvals from the Cameroonian Ministry of Scientific Research and Innovation (Ref. 015/MINRESI/B00/C00/C10/C14) and Ministry of Livestock, Fisheries and Animal Industries (Ref. 061/L/MINEPIA/SG/DREPIA/CE) were also granted. This study was further placed on record with the South African National Department of Agriculture, Forestry and Fisheries [Reference: 12/11/1/5 (878)].
4.3. Sampling Procedures and Survey
4.3.1. Animal Sampling Procedure
Apparently healthy and freshly slaughtered/stunned pigs were randomly sampled in both Cameroon and South Africa. The interior cavity of both anterior nares were swabbed and rectal swabs of pigs were obtained using sterile Amies swabs (Copan Italia Spa, Brescia, Italia). Altogether, 432 nasal and rectal pigs were collected in both countries, with the number of samples from each slaughterhouse (SH001, n = 129; SH002, n = 57; SH003, n = 30; SH004, n = 120; SH005, n = 96) proportional to the annual pig production per site.
4.3.2. Human Sampling Procedure
Total sampling was employed where all exposed workers (≥21 years old) willing to participate were recruited in the study upon oral and written informed consent. Participants were requested to answer a questionnaire describing socio-demographic and medical/clinical history, as well as probable risk factors associated with ESBL-PE emergence/colonization and spread. Amies swab was used to collect both anterior nares and hand (between fingers for each right and left hand) samples which were processed within 4 h of collection.
4.4. Bacteriological Analysis
For the bacteriological analysis, three individual pig samples were pooled per abattoir, gender, specimen and area of breeding leading to 288 pools (144 nasal and 144 rectal) representing 432 original specimens collected from 432 pigs. Pooled pig samples and human swabs were cultured onto an in-house selective 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).
4.5. ESBL Detection, Species Identification and Antimicrobial Susceptibility Testing
Each colony with a unique morphotype growing on MCA+CTX was screened for ESBL production through the standard double disk synergy test (DDST) as recommended by the Clinical Laboratory and Standards Institute (CLSI) [
16].
A panel of 19 antibiotics including amoxicillin + clavulanic acid, ampicillin, cefuroxime, cefuroxime axetil, cefoxitin, cefotaxime, ceftazidime, cefepime, imipenem, ertapenem, meropenem, amikacin, gentamicin, ciprofloxacin, tigecycline, piperacillin/tazobactam, nitrofurantoin, colistin and trimethoprim-sulfamethoxazole, were tested using Vitek
® 2 System and Vitek
® 2 Gram Negative Susceptibility card (AST-N255) (BioMérieux, Marcy l’Etoile, France). The CLSI was used for interpretation of the results excepted for colistin, piperacillin/tazobactam, amoxicillin + clavulanic acid and amikacin that were interpreted using EUCAST breakpoints [
17].
E. coli ATCC 25922 was used as the control.
4.6. Genotypic Relatedness Determination of ESBL-Producing Escherichia coli
The Thermo Scientific
® GeneJet Genomic DNA purification kit (Thermo Fisher Scientific, Johannesburg, South Africa) was used for the genomic DNA extraction following the manufacturer’s instructions. ERIC-PCR was carried out using the primers ERIC 1 5′-ATG TAA GCT CCT GGG GAT TCA C-3′ and ERIC 2 5′-AAG TAA GTG ACT GGG GTG AGC G-3′ [
18]. Reactions were performed in a 10 µL final solution containing 5 µL Dream
Taq Green Polymerase Master Mix 2× (Thermo Fisher Scientific, South Africa), 2.8 µL nuclease free water, 0.1 µL of each primer (100 μM) and 2 µL DNA template and run in an Applied Biosystems 2720 programmable thermal cycler (Thermo Fisher Scientific, Johannesburg, South Africa). The ERIC-PCR protocol implemented included 3 min of initial denaturation at 94 °C, followed by 30 cycles consisting of a denaturation at 94 °C for 30 s, annealing at 50 °C for 1 min, extension at 65 °C for 8 min, a final extension at 65 °C for 16 min and final storage at 4 °C. ERIC profiles were digitized and analysed using Bionumerics (version 7.6, Applied Maths, Austin, TX, USA). The similarity between each strain was assessed using Dice coefficient and dendrograms were constructed using the Unweighted Pair-Group Method Algorithm (UPGMA).
4.7. Data Analysis
Data was encoded and entered into Epi Info (version 7.2, CDC, Atlanta, GA, USA) and Excel (Microsoft Office 2016) and analysed using STATA (version 14.0, STATACorp LLC, College Statioon, TX, USA). A data set was designed for specific human results and, combined animal and abattoir data. Abattoirs were classified as ESBL-positive if an ESBL-PE was identified from at least one pool (nasal or rectal samples). Likewise, each human was categorized as carrier or non-carrier, with carrier being defined as having ESBL-PE in at least one site (nares or hand).
The ESBL-PE prevalence was compared between categories with the chi square test (p < 0.05). The relationship between ESBL-PE carriage in pigs and humans was ascertained using logistic regression analyses adjusted for clustering at abattoir level. Likewise, risk factors for ESBL-PE carriage were determined univariately and selected for multivariate analysis when the p-value was <0.2. The McFadden’s pseudo R2 statistic (maximum likelihood method) was used to check the model fit and the final model included all determinants for which the pseudo R2 was the most elevated with p < 0.05 for each dependent variable.