GENERAL INTRODUCTION
III. Literature review
3. Study design and methodology
Between March and October 2016, nasal and rectal swabs of pigs and nasal and hand swabs of occupationally exposed workers older than 21 years old were collected in five abattoirs in both Cameroon and South Africa. All samples were screened on selective media (MacConkey agar and Mannitol salt agar) supplemented with antibiotics (cefotaxime and cefoxitin) and identification was undertaken by colony morphology, catalase and oxidase tests, with confirmation by the Vitek® 2 System (BioMérieuX, Marcy l’Etoile, France). The cefoxitin screening and double disk synergy tests were used for methicillin resistance and extended- spectrum beta-lactamase production in staphylococci and Enterobacteriaceae, respectively.
Repetitive-palindromic polymerase chain reaction (REP-PCR) and enterobacterial-repetitive- polymerase chain reaction (ERIC-PCR) were used to ascertain the clonal relatedness of MRSA and ESBL-PE isolates, respectively. Genomic DNA of representative isolates were further subjected to whole genome sequencing (WGS) using an Illumina MiSeq platform to delineate the molecular epidemiology, resistance genes, virulence factors, phage-related regions and circulating clonal lineages of these isolates. Generated reads were assembled with CLC Genomics Workbench and SPAdes, annotated with PGAP and RAST. ResFinder, VirulenceFinder, PlasmidFinder and PHAST were used to identify antibiotic resistance genes, virulence factors, plasmids and phage associated-regions. The multilocus sequence type (MLST) of the isolates was determined from the WGS data. Whole genome phylogenetic analysis was performed using the Rapid large-scale prokaryote pan genome analysis (Roary).
37 4. Thesis outline
The present study is outlined in the form of published journal articles and unpublished manuscripts, and is delineated by the following seven chapters:
▪ Chapter 2. Article I: Antibiotic Resistance in the Food Chain: A Developing Country- Perspective. Front Microbiol. 7:1881. doi: 10.3389/fmicb.2016.01881. This review evidences the danger of ABR in the food chain and particularly in developing countries as a serious global public health threat by highlighting the transmission routes of ABR along the food chain and comparing the status of ABR in food animals in developed vs.
developing countries. Prevention and containment measures of ABR from farm-to-fork are also delineated. This paper addresses objectives One and Ten.
▪ Chapter 3. Article II: Antibiotic Resistance in Food Animals in Africa: A Systematic Review and Meta-Analysis. This systematic review analysed the published literature on the emergence/prevalence of ABR in food animals in Africa, in order to (1) describe the distribution of ABR-bacteria in food animals; (2) highlight the need to restrict the use of antibiotics in agriculture, and (3) provide evidence to follow the One Health approach to contain the emergence and spread of ABR on the continent. This manuscript addresses objectives Two and Ten. It has been submitted to Microbial Drug Resistance and is currently under review.
▪ Chapter 4. Article III: Emergence and Spread of Extended Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae in Pig Slaughterhouses and Exposed Workers: A Multicentre Comparative Study Between Cameroon and South Africa. This original research article reports on and compares the prevalence, risk factors as well as clonal relatedness of ESBL-PE circulating in Cameroonian and South African slaughterhouses and addresses objectives Three, Four, Five, Six, Seven, Nine and Ten. It has been submitted to PloS One and currently under review.
▪ Chapter 5. Article IV: Mannitol-Fermenting Methicillin-Resistant Staphylococci (MRS) in Pigs and Abattoir Workers in Cameroon and South Africa: A Serious Food Safety Threat.
It has been submitted to the International Journal of Food Microbiology and is currently under review. It reports on and compares the prevalence, risk factors as well as clonal relatedness of MRS circulating in Cameroonian and South African slaughterhouses and addresses objectives Three, Four, Five, Six, Seven, Nine and Ten.
▪ Chapter 6. Article V: Genome Analysis of Methicillin-Resistant Staphylococcus aureus Isolated from Pigs: Emergence of the Clonal Lineage ST398 in Cameroon and South Africa. This original research article has been submitted to Frontiers in Microbiology and
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is currently under review. It reports on antibiotic resistance genes, virulence factors, mobile genetic elements and genetic lineages of circulating MRSA strains isolated from pigs in Cameroonian and South African abattoirs using whole genome sequencing (WGS) and addresses objectives Eight and Nine. This paper will reference “Mannitol-Fermenting Methicillin-Resistant Staphylococci (MRS) in Pigs and Abattoir Workers in Cameroon and South Africa: A Serious Food Safety Threat” which is expected to be published first.
▪ Chapter 7. Article VI: Whole Genome Sequencing of Circulating ESBL-producing Klebsiella pneumoniae Isolated from Pigs and Abattoir Workers in Cameroon. This original research article has been submitted to Frontiers in Microbiology and is currently under review. It reports on antibiotic resistance genes, virulence factors, mobile genetic elements, and genetic lineages of circulating ESBL-producing K. pneumoniae strains isolated in Cameroonian slaughterhouses using whole genome sequencing and addresses objectives Eight and Nine. This paper will reference the “Emergence and Spread of Extended Spectrum Beta-Lactamase (ESBL)-Producing Enterobacteriaceae in Pig Slaughterhouses and Exposed Workers: A Multicentre Comparative Study Between Cameroon and South Africa” paper, which is expected to be published first.
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Chapter 8. Conclusion: This chapter presents the extent to which the overarching aim was met by reviewing the key findings for each specific objective. It further delineates the significance, limitations and recommendations of the study for the field of food safety and antibiotic resistance.39