Genomic analysis of antimicrobial resistant escherichia coli, enterococcus and salmonella species isolated from humans and broiler chickens in Lusaka and Copperbelt provinces in Zambia.

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Yamba, Kaunda
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The University of Zambia
Antimicrobial resistance (AMR) is a global public health and economic threat involving humans, animals and the environment. Globally, AMR threatens historic gains attained in the treatment of infectious diseases. Escherichia coli and Enterococcus species initially considered harmless members of the gut resident biota, are now important human pathogens associated with a wide range of clinical syndromes, including urinary tract infections (UTIs) and bloodstream infections (BSIs). Salmonellae are the leading cause of community acquired BSIs in low- and middle-income countries (LMIC). In order to understand AMR, the distribution and the genomic relatedness of these pathogens, a holistic approach in the three sectors is of critical importance. The aim of this study was to determine the phenotypic and genotypic characteristics of E. coli, Enterococcus and Salmonella species and establish genotypic relatedness of E. coli and Enterococcus species isolated from humans (clinical and carriage) and poultry in Lusaka and Copperbelt provinces of Zambia. This was a two-tier cross-sectional study comprised of human and poultry bacterial isolates. The human study had two components: hospitalized patients and the healthy community. E. coli and Enterococcus species were isolated from hospitalised patients with UTIs and BSIs, healthy pregnant women and children < 5 years and from market-ready-chickens while Salmonella species was isolated from hospitalised patients with BSIs only. Antimicrobial susceptibility patterns were determined by Kirby-Bauer disc diffusion method and the Vitek 2 compact automated machine. AMR data was entered into Excel spreadsheets and WHOnet 2020, then analysed in STATA version 14. Subsets of E. coli, Enterococcus and Salmonella species were selected for whole genome sequencing (WGS) using Illumina short read sequencing. The raw sequence data of the three pathogens were uploaded and investigated on different platforms such as EnteroBase and SnapperDB. Assembled sequence data for all three pathogens were analysed using online bioinformatics pipelines at the Centre for Genomic Epidemiology (GCE). Phylogenetic analysis of E. coli and Salmonella enterica were carried out on Enterobase and visualised using Microreact. High levels of resistance to a number of antibiotic classes were recorded in all the three studied pathogens. Notably, resistance to third generation cephalosporins and extended spectrum betalactamases in E. coli clinical isolates was 62% and 89% respectively. It was interesting to note the presence of similar resistance gene determinants and incompatibilty (Inc) plasmid replicons FII, FIB, FIA and I1 in both clinical and non-clinical E. coli strains. The most prevalent sequence type (ST) was E. coli-ST131(79%), most of which were clinical isolates (97%). Notable was the wide diversity of other STs in the healthy community and poultry, most of which were phylogenetically unrelated but carried similar resistance genes on similar plasmids. E. faecalis (52%) was the most prevalent Enterococcus species with emerging resistance to ampicillin/penicillin (26%/34%), high-level gentamicin (12%) and quinupristin-dalfopristin (1%) in clinical isolates. Salmonella infections were mostly caused by genomic diverse S. Typhi (86%) with an emergence of S. Typhimurium ST313 (3%). Salmonella infections recorded an increase in resistance to ciprofloxacin (1% to 20%) and ceftriaxone (1% to 11%) in 2020/2021 compared to 2018/2019. All the three pathogens had phylogroups/STs that are known to be commensals or less virulent strains but associated with clinical infections. The occurrence of strains known to be commensals as causative agents of BSIs/UTIs, the distribution of similar antimicrobial resistance profiles and resistance gene determinants on the same type of transferable plasmids in all the three interfaces, supports the possibility of cross transmission across species. It further supports the ability of previously non-pathogenic strains to acquire traits that allow them to become pathogenic and further spread AMR. These findings warrant for adoption of policies and regulations that address the prevention and containment of AMR using the one health approach.
Thesis of Doctor of Philosophy in Public Health (One Health).