Epidemiology of antimicrobial resistance at the livestock-human interface in an urban environment: a One Health approach

Livestock have been implicated as a reservoir for antimicrobial resistant (AMR) bacteria that may spread to humans, with the keeping of livestock widely postulated as a risk factor for AMR in humans. However, quantitative evidence of the role of livestock in the emergence and transmission of AMR bacteria to human populations is lacking. This thesis focuses on the role of livestock keeping as a potentially high-risk interface for AMR transmission between humans and livestock in urban Nairobi. To achieve this, E. coli isolates were systematically collected from sympatric human and livestock populations in 99 households across Nairobi, Kenya. E. coli was characterised both phenotypically (through antimicrobial susceptibility testing) and genetically (through whole genome sequencing). In the first part of this thesis, I conduct a comprehensive systematic review to investigate existing evidence that food animals are responsible for transfer of resistant E. coli and their AMR determinants to humans. I demonstrate that the current evidence regarding transmission of drug resistance between food animals and humans is limited and that similarity of AMR bacteria or AMR determinants in the two populations does not, by itself, provide information on directionality of transfer. I highlight the need to use high resolution genomic analysis on human and livestock bacterial samples collected in time and space to better understand the direction and frequency of AMR transmission between these populations. Next, utilising AMR phenotypes and genotypes, I explored the variation in carriage of AMR E. coli and investigated the role of livestock ownership as a risk factor for AMR carriage in humans. First, I explored the epidemiology of clinically relevant AMR phenotypes and AMR genetic markers. I detected high rates of AMR phenotypes, with 47.6% and 21.1% of isolates displaying resistance to ≥ 3 and ≥5 antimicrobial classes respectively. Whole-genome sequencing revealed 60 acquired genes and 14 point mutations conferring AMR to 9 antimicrobial classes. sul2, strA, strB, tetA, and blaTEM-1B were the most frequently detected AMR genes conferring resistance to sulfonamides, aminoglycosides, tetracyclines, and β-lactams respectively – the most commonly found phenotypes. Highest carriage of AMR genes and phenotypes was observed in humans, pigs and poultry compared to goats, rabbits and bovines. Secondly, I demonstrated that the presence of livestock in the household did not influence phenotypic or genotypic AMR carriage in humans, but the impact of keeping livestock on human AMR carriage was instead influenced by presence of animal manure in the household. Utilising high resolution sequencing data, I proceeded to investigate the patterns of bacterial relatedness and strain sharing as a proxy for transmission potential. I showed that livestock and human isolates are genetically heterogeneous, with minimal evidence of clustering by host group, and that E. coli genomes in humans did not segregate according to livestock ownership. Next, I found evidence of 91 sharing events differing by less than ten base pairs (59 involving livestock isolates only 23 human isolates only, and 9 between humans and livestock), and that most of the sharing events were confined within households with only occasional instances of spread between household. I also demonstrate that high-resolution sequence-based analysis of SNPs is more discriminatory than MLST – a widely used tool in describing transmission of E. coli. Next, I described the patterns of antimicrobial sales in humans and livestock, and the level of awareness and common behaviours related to antimicrobial prescribing amongst human and veterinary pharmacists in urban Nairobi. β- lactams, fluoroquinolones, first and second generation cephalosporins, and metronidazole were the most commonly purchased human antimicrobials while tetracyclines, sulphonamides, penicillins, and macrolides were the most commonly purchased veterinary antimicrobials. This finding was in line with the resistance phenotypes and genotypes described in this thesis. I found that whilst most pharmacists were knowledgeable about antimicrobial use and AMR, inappropriate prescribing practices were common and that over the counter sale of antimicrobials, without a prescription, was a common occurrence in both human and veterinary drug stores. In the final section of the thesis, I investigated the co-occurrence patterns of acquired AMR genes and the role of conjugative plasmids on the epidemiology of AMR spread. I found evidence of co-location of multiple AMR genes in both human and livestock isolates, potentially enabling acquisition and dissemination of multi-drug resistance phenotypes in a single step. I found a diversity of known plasmids and plasmid replicons that were associated with the distribution of acquired AMR genes. To conclude, I discuss the findings of this thesis in the context of the current epidemiology of AMR pathogens at the human-livestock interface and highlight future directions for research on AMR transmission, and discuss implications of my findings for public health. This thesis demonstrates how fine-scale genomic analysis explicitly embedded within an epidemiologically structured sampling framework can be utilized to track bacterial sharing and in the surveillance of AMR prevalence in a low income urban setting. The connectivity of bacteria and their AMR determinants between humans and livestock and the ultimate impacts upon human health lends strong support for a holistic ‘One Health’ perspective for AMR surveillance

Co-infection of pigs with Taenia solium cysticercosis and gastrointestinal parasites in Eastern and Western Uganda

A study was carried out in Kamuli and Hoima districts in Eastern and Western regions of Uganda to determine the Taenia solium porcine cysticercosis (PCC) and gastrointestinal (GI) parasites co-infection status in pigs. One hundred sixty-one households were selected randomly and visited between November and December 2019. A household questionnaire was administered, and faecal and blood samples were collected from at least one pig older than 3 months per household. A blood sample was obtained from a jugular venipuncture, and a rectal faecal sample was obtained. Taenia spp. circulating antigen levels in the sample sera were tested using a commercial enzyme-linked immunosorbent assay kit, apDia™ cysticercosis Ag ELISA. The modified McMaster technique was used to identify and quantify the GI parasites. The apparent animal-level seroprevalence for PCC was 4.8% (95% CI 2.7–7.1) and differed across the two districts (p = 0.018). At the pig herd level, the prevalence was 9.7% (95% CI 5.5–14.4). The prevalence of the different nematode eggs and coccidian oocysts in the two districts was as follows: strongyles 79.0% (95% CI 74.3–83.6), coccidia 73.3% (95% CI 68.3–78.6), Trichuris spp. 7.4% (95% CI 4.9–10.6), Strongyloides ransomi 2.1 (95% CI 0.7–3.5) and Ascaris spp. 4.9 (95% CI 2.8–7.4). Overall, across the two districts, the arithmetic mean for the oocysts per gram (OPG) for coccidia was 2042.2 ± 5776.1, and eggs per gram (EPG) were the highest in strongyles 616.1 ± 991. Overall, 57.4% of the porcine cysticercosis seropositive pigs were also positive for at least one of the gastrointestinal helminths which included strongyles, Strongyloides ransomi, Trichuris spp. and Ascaris spp. The co-infection status of pigs with both PCC and GI parasites demonstrated by this study can provide an incentive for integrating the control and management of both parasites with oxfendazole. Further studies are required to understand the feasibility of using oxfendazole including cost–benefit analysis and the acceptability by local stakeholders for the control of T. solium cysticercosis and gastrointestinal parasites in pigs

Value chain analysis as a tool for assessing food safety risks in the Nairobi pork food system

In Nairobi, with 3.1 million consumers and 30,000 pigs, the pork system may represents a major source of zoonotic pathogens. Yet, this system and its public health risks have not been described. The study used value chain analysis, a well-known method in economics, to investigate zoonosis and food safety risks practices in the Nairobi pork food system. A cross-sectional study of the Nairobi pork system collected data through 25 focus group discussions and 436 individual interviews with farmers, traders, abattoir owners, large companies’ managers, retailers, government officers and consumers. Data were analysed to identify, describe and quantify the main pork chain profiles, their associated zoonosis and food safety risks practices and their link to governance, the distribution of benefits and barriers to improving the system. Six pork chain profiles were identified with the ‘large integrated company’ profile accounting for 62% of pork marketed through abattoirs. Pigs in slums were channelled directly to consumers and butchers or through less integrated markets. Main zoonosis and food safety risk practices for city pig keepers were: handling and consumption of sick pigs; and swill and scavenging feeding. In less integrated abattoirs these risks were: lack of traceability, cold chain systems and adequate cleaning and sterilising practices and equipment. For the retailers, there was a lack of hygiene linked to poor infrastructure, scarcity of water and cleaning practices. Large companies govern the high end market for pork where barriers to improvements were less. In the lower end poor profit margins and unequal benefit distribution led to issues on around investments in infrastructure, cold chains and human capacity building plus difficulties with meeting feeding and animal health costs. Conclusions This study identified the main zoonoses and food safety risk practices and the people involved in risk taking activities to help future control programmes in the Nairobi pork system. The integration of value chain and analysis of risks practices proved useful and represent the way forward for epidemiologist working in developing countries

Detection of circulating Antigens for Taenia spp. in pigs slaughtered for consumption in Nairobi and surroundings, Kenya

Taenia solium a zoonotic tapeworm, responsible for neurocysticercosis in humans is a major public health threat, being a leading cause of acquired epilepsy in endemic regions. Eastern and southern African nations have experienced a recent rapid growth in pig production, including small-scale, free-range systems, with an accompanying increased risk of T. solium transmission. Seven hundred blood samples were collected from randomly selected pigs presented for slaughter at one of the largest porcine abattoir supplying unprocessed pork to Nairobi city and its surroundings. The samples were tested using an antigen ELISA to determine the prevalence of infection with Taenia spp

The sero-epidemiology of Coxiella burnetii (Q fever) across livestock species and herding contexts in Laikipia County, Kenya

Coxiella burnetii, the causative agent of Query fever (Q fever), is among the most highly infectious zoonotic pathogens transmitted among livestock, with chronic effects challenging to veterinary and medical detection and care systems. Transmission among domestic livestock species can vary regionally due to herd management practices that determine which livestock species are raised, whether or not livestock are in contact with wildlife, and the susceptibility of these livestock to infection. To explore how different livestock management practices are associated with the risk of infection in multispecies environments, we carried out a comparative study of three types of herd management systems in the central Kenyan county of Laikipia: agro‐commercial, mixed conservancy/commercial, and smallholder ranches. We tested C. burnetii antibody seroprevalence in four common livestock species. Across all management types, the highest seroprevalence was in camels (20%), followed by goats (18%), sheep (13%), and cattle (6%). We observed a lower odds of testing seropositive for young compared to adult animals (adjusted OR = 0.44 [95% CI 0.24, 0.76]), and for males compared to females (adjusted OR = 0.52 [95% CI 0.33, 0.80]). Animals from mixed conservancy/commercial and smallholder operations had a higher odds of testing seropositive compared to animals from agro‐commercial ranches (adjusted OR = 5.17 [95% CI 2.71, 10.44] and adjusted OR = 2.21 [95% CI 1.17, 4.43] respectively). These data suggest that herd management practices might affect the transmission dynamics of C. burnetiiin arid African ecosystems like those seen in Kenya where several transmission modes are possible, risk of drought has promoted new livestock species such as camels, and multiple wildlife species may co‐occur with livestock on the landscape. Further longitudinal studies are needed to disentangle the mechanisms underlying these patterns, and further explore transmission patterns between wildlife, domestic animal, and human populations

Antimicrobial use and antimicrobial resistance in broiler farms in peri-urban Wakiso, Uganda

Objective: A cross sectional study was carried out to 1) describe the antimicrobial use patterns among poultry farmers in Wakiso 2) establish resistant profiles of Salmonella, E. coli and Enterococci among poultry farms in Wakiso. Methods: A cross sectional study in Wakiso district was carried out in October and November 2021 for a period of 6 weeks. A semi structured questionnaire was used to collect data on antimicrobial use on the farms. Boot sock sampling was used to collect samples from the whole poultry house. A composite sample of faecal material was also collected from the poultry house. The information on antimicrobial use is being analyzed for descriptive statistics. The faecal samples were cultured in the laboratory for Isolation of Salmonella, E. coli and Enterococci. The Isolates will further be tested to establish their resistant profiles. The composite sample was used to quantify resistant coliforms against cefotaxime and colistin Results: A total of 200 poultry farms in Wakiso were visited, 211 boot sock samples and 211 composite samples were collected. 29.85% of the respondents were male while 70.15% were female.37.38% had attained secondary level of education. There was observed widespread use of antimicrobials on the farms for treatment of different conditions as well as prevention of disease. The most used groups of antibiotics were, Penicillins, Tetracyclines and Aminoglycosides while Sulphonamides were the least used. From the laboratory analysis, out of the 211 samples collected, Salmonella was isolated at a rate of 8.5%, E. coli at 77.3% and Enterococcus at 62.6%. Resistant coliforms to cefotaxime (5I.7%) and colistin (45.5%) were also identified. Conclusion: There are indications of resistant coliforms among poultry farms in Wakiso. This is attributed to the observed continuous use of antibiotics on the poultry farms. There is need to improve antimicrobial use among farmers. Proper diagnosis of conditions is one possible way that will reduce the use of different antibiotics at farm level

Socio‐ecological drivers of vertebrate biodiversity and human‐animal interfaces across an urban landscape

Urbanization can have profound impacts on the distributional ecology of wildlife and livestock, with implications for biodiversity conservation, ecosystem services and human health. A wealth of studies have assessed biotic responses to urbanization in North America and Europe, but there is little empirical evidence that directly links human activities to urban biodiversity in the tropics. Results from a large-scale field study conducted in Nairobi, Kenya, are used to explore the impact of human activities on the biodiversity of wildlife and livestock with which humans co-exist across the city. The structure of sympatric wildlife, livestock and human populations are characterized using unsupervised machine learning, and statistical modelling is used to relate compositional variation in these communities to socio-ecological drivers occurring across the city. By characterizing landscape-scale drivers acting on these interfaces, we demonstrate that socioeconomics, elevation and subsequent changes in habitat have measurable impacts upon the diversity, density and species assemblage of wildlife, livestock and humans. Restructuring of wildlife and livestock assemblages (both in terms of species diversity and composition) has important implications for the emergence of novel diseases at urban interfaces, and we therefore use our results to generate a set of testable hypotheses that explore the influence of urban change on microbial communities. These results provide novel insight into the impact of urbanization on biodiversity in the tropics. An understanding of associations between urban processes and the structure of human and animal populations is required to link urban development to conservation efforts and risks posed by disease emergence to human health, ultimately informing sustainable urban development policy